Particle analysis assay for biomolecular quantification

ABSTRACT

A method is provided for carrying out multi-step separations of objects bearing at least two binding sites. In the first step, a first binder/bead composition is bound to objects that bear the first binding site, and then unbound objects, i.e. objects not bearing the first binding site, are separated from bound objects. In the second step, a second binder/bead composition is bound to the remaining objects that bear the second binding site, and then the objects that are bound to both beads are removed from those objects that are bound to only one bead. The beads can differ in magnetic responsiveness, charge, size, color, and the like, and these differences can be used to carry out the separation steps.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of application Ser. No. 10/089,560, filedApr. 1, 2002, now U.S. Pat. No. 6,994,971 which was the National Stageof International Application No. PCT/US00/27883, filed Oct. 10, 2000,which claims the benefit of U.S. Provisional Application No. 60/158,664,filed Oct. 8, 1999, which applications are incorporated herein by thisreference.

BACKGROUND OF THE INVENTION

The present invention relates to methods for isolation, separation, anddetection of selected objects. More particularly, the invention relatesto methods for performing separations of objects, such as, withoutlimitation, nucleic acids, proteins, cells, organelles, and the like.

Separations of biological objects, such as proteins, chromosomes,nucleic acids, cells, organelles, and the like, and other types ofobjects are important in various detection, isolation, quantification,and diagnostic processes. Specificity and sensitivity are two importantparameters that are generally desired in these separation schemes.

Hybridization probes are widely used to detect and/or quantify thepresence of a particular nucleotide sequence in a mixed sample ofnucleotide sequences. Hybridization probes detect the presence of aparticular nucleotide sequence, referred to herein as a target sequence,through the use of a complementary nucleotide sequence that selectivelyhybridizes to the target nucleotide sequence. For a hybridization probeto hybridize to a target sequence, the hybridization probe must containa nucleotide sequence that is complementary to the target sequence. Thecomplementary sequence must also be sufficiently long for the probe toexhibit selectivity for the target sequence over non-target sequences.

Hybridization assays can be designed to detect the presence or absenceof a particular nucleotide sequence, for example the presence of a genein a DNA sequence. Hybridization assays can also be designed to detectthe movement of a nucleotide sequence relative to another nucleotidesequence in a sample, for example the presence of a gene on a chromosomethat is known to be normally located on a different chromosome, e.g.,the detection of the abl gene on chromosome 22 in human leukemiapatients (e.g., Tkachuk et al., 250 Science 559-562 (1990); C-TRAKtranslocation detection system commercially available from Oncor, Inc.,Gaithersburg, Md.; U.S. Pat. No. 5,447,841; U.S. Pat. No. 5,731,153; andU.S. Pat. No. 5,783,387).

As used herein, “nucleotide sequence aberrations” refers torearrangements between and within nucleic acids, particularlychromosomal rearrangements. “Nucleotide sequence aberrations” alsorefers to the deletion of a nucleotide sequence, particularly chromosomedeletions. As used herein, the term “nucleic acids” refers to both DNAand RNA.

A chromosome translocation is an example of a nucleotide sequenceaberration. A chromosome translocation refers to the movement of aportion of one chromosome to another chromosome (inter-chromosomerearrangement), as well as the movement of a portion of a chromosome toa different location on that chromosome (intra-chromosomerearrangement). In general, chromosome translocations are characterizedby the presence of a DNA sequence on a particular chromosome that isknown to be native to a different chromosome or different portion of thesame chromosome. Because chromosome translocations involve the movementof a nucleotide sequence within a sample, as opposed to the appearanceor disappearance of the nucleotide sequence, it generally is notpossible to detect a chromosome translocation merely by assaying for thepresence or absence of a particular nucleotide sequence.

Chromosome translocations are known to increase in frequency uponexposure to radiation and certain chemicals. Measurement of thefrequency of chromosome translocations after exposure to radiation or aparticular agent is therefore useful for evaluating the tendency of suchagents to cause or increase the frequency of chromosome translocations.Also, the frequency (translocations per cell) of chromosometranslocations measured in blood lymphocytes from an individual can beused as a quantitative measure of the amount of prior exposure to suchagents (e.g., T. Straume and J. Lucas “Validation studies for monitoringof workers using molecular cytogenetics,” Biomarkers in OccupationalHealth: Progress and Perspectives (M. L. Mendelsohn, J. P. Peeters, andM. J. Normandt, Eds.), Joseph Henry Press, Washington D.C., pp. 174-193(1995)).

Chromosome translocations are also known to be associated with specificdiseases, including, for example lymphomas and leukemia, such asBurkitt's lymphoma, chronic myelocytic leukemia, chronic lymphocyticleukemia, and granulocytic leukemia, as well as solid tumors such asmalignant melanoma, prostate cancer, and cervical cancer. A method forefficiently detecting a translocation associated with a disease isneeded as a method for diagnosing disease, follow-up of cancer therapypatients, research, and population studies.

Fluorescence in situ hybridization (FISH) using chromosome-specificcomposite hybridization probes (“chromosome painting”) was developed asan assay for detecting chromosome translocations. FISH and selectedapplications of the FISH method are described in Pinkel et al., 83 Proc.Nat'l Acad. Sci. USA 2934-2938 (1986); Straume et al., UCRL 93837(1986); Pinkel et al., 85 Proc. Nat'l Acad. Sci. USA 9138-9142 (1988);U.S. Pat. No. 5,447,841; Lucas et al., 62 International Journal ofRadiation Biology 53-63 (1992); Straume et al., 62 Health Physics122-130 (1992); Straume and Lucas, 64 Int. J. Radiat. Biol. 185-187(1993).

The fluorescent hybridization probes used in FISH-based chromosomepainting are substantially chromosome-specific, i.e., they hybridizeprimarily to a particular chromosome type. Unique or substantiallyunique probes may be used to limit non-specific hybridization. Adiscussion of so-called unique, middle repetitive, and highly repetitivesequences and their implications for hybridization probes is found inU.S. Pat. No. 5,447,841. Chromosome translocations are identified in theFISH assay by visually scanning individual cells for the presence of twodifferent fluorescent signals on a single chromosome, the twofluorescent signals originating from two different cocktails of FISHprobes, each probe cocktail having homology to a different chromosometype.

Because each FISH probe hybridizes to a specific chromosome type and notto the chromosome translocation itself, it is not possible to determinethe frequency of chromosome translocations directly from thefluorescence signal emanating from a FISH probe. Rather, the frequencyof chromosome translocations in a cell sample must be determinedaccording to FISH assays by visually scanning individual metaphase cellson slides and identifying whether the two fluorescent signals appear onthe same chromosome. The need to visually scan such individual cellseffectively limits the number of cells that can be assayed, therebyreducing the sensitivity of the FISH assay, introducing the possibilityof human error, and greatly increasing cost per analysis.

Accordingly, a fast, accurate method is needed for quantifyingchromosome translocations and other nucleotide sequence aberrations. Inparticular, a method is needed that can isolate and quantify nucleotidesequence aberrations contained in the nucleic acid of a sample of cellswithout the need to analyze each cell individually.

U.S. Pat. No. 5,731,153 relates to a two-step separation procedure thatuses two solid supports, each coated with unique complexing agents thatbind to hybridization probes complementary to different targetsequences. This procedure requires detachment of the target sequencefrom the first solid support after the first separation step andreattachment of the target sequence to a second solid support before thesecond separation step can be performed. The requirement forre-attachment of the target sequence is particularly problematic andwould add significantly to the complexity and cost of commercialseparation kits using such methodology and reduce the precision of theassay because of variability in the detachment/reattachment step.Further, this procedure is limited to two types of solid supports, butit would be useful to have more support options to facilitate multiplesimultaneous analyses. Moreover, the preferable methods forquantification described in U.S. Pat. No. 5,731,153 require either veryexpensive and uncommon equipment (e.g., measure ¹⁴C by accelerator massspectrometry) or much less quantitative methods such as the detection offluorescence labels on reporter nucleic acid probes. Also, the method inU.S. Pat. No. 5,731,153 is limited to separation of nucleic acids,whereas it would be advantageous to separate other types of objects aswell.

Unfortunately, methods available for the quantification of chromosomalrearrangements are either very costly and inefficient, e.g.,cytogenetic-type analyses (H. J. Evans et al., 35 Chromosoma 310-325(1971); D. Pinkel et al., 83 Proc. Natl. Acad. Sci. USA 2934-2938(1986); D. Pinkel et al., 85 Proc. Natl. Acad. Sci. USA 9138-9142(1988); D.C. Tkachuk et al., 250 Science 559-562 (1990)), or requiresmall sequences such as fusion mRNAs that may be amplified by PCR anddetected (M. H. Delfau et al., 4 Leukemia 1-5 (1990); A. Zippelius & K.Pantel, 906 Annals NY Acad. Sci. 110-123 (2000)). Cytogenetics require ahighly trained technician to visually score metaphase or interphasecells using a microscope and make judgements about what is observed. PCRis less labor intensive than cytogenetics but is of limited utility indirect DNA-based detection of most chromosomal translocations becausethe fusion points tend to be variable. D.C. Tkachuk et al., 250 Science559-562 (1990); E. Solomon et al., 254 Science 1153-1160 (1991). Theselimitations have essentially restricted PCR to the detection of fusionmRNAs, which may not always be known, may arise from ectopic expression,or may be expressed deficiently (A. Zippelius & K. Pantel, 906 Annals NYAcad. Sci. 110-123 (2000)).

In view of the foregoing, it will be appreciated that providing aseparation method that does not require reattachment of the targetsequence to a solid support to perform the second step, does not requirePCR, is highly quantitative, can be accomplished using readily availablelaboratory equipment, can be used for multiple simultaneous analyses,and that is applicable to the isolation and quantization of manydifferent kinds of objects, including nucleic acids, metaphasechromosomes, proteins, cells, organelles, and the like, would be asignificant advancement in the art.

Such methods are disclosed herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods for separation and quantizationof objects, including nucleic acids, chromosomes, proteins, organelles,cells, and the like.

In one preferred embodiment, the present invention relates to a methodfor separating nucleotide sequence aberrations from normal nucleotidesequences and quantification of the frequency of abnormal sequences. Asused herein, “nucleotide sequence aberration” refers to rearrangementsbetween and within nucleotide sequences, particularly chromosomes.“Nucleotide sequence aberration” also refers to the deletion of anucleotide sequence, particularly chromosome deletions. As used herein,the term “nucleic acids” refers to both DNA and RNA of any origin andany level of organization, e.g., DNA, chromatin, and chromosome.

A method is provided for separating and quantifying nucleic acids thatinclude a nucleotide sequence aberration, the nucleotide sequenceaberration being identified by the presence of nucleotide sequences thatinclude a first, a second, and additional ones if desired, nucleotidesequence types.

According to a preferred embodiment of the present invention, anucleotide sequence aberration is isolated by separation of nucleicacids having both a first nucleotide sequence type (e.g., from a firstchromosome) and a second nucleotide sequence type (e.g., from a secondchromosome) from nucleic acid sequences not having both first and secondsequence types. The presence of the first and the second nucleotidesequence types on the same nucleic acid indicate the presence of anucleotide sequence aberration. Thus, nucleic acids that contain anucleotide sequence aberration, characterized by their having nucleicacid sequences of both a first and a second nucleic acid sequence type,are selectively isolated. Once isolated, these sequences may bedetected, quantified, and/or characterized.

In an illustrative embodiment of the invention, a target nucleic acid isisolated from a mixture of nucleic acids in a sample by hybridizing twoor more hybridization probes to the mixture of nucleic acids, each probetype being specific for non-overlapping sequences on the target nucleicacid and containing complexing agents specific for selected types ofsolid support surfaces. Preferred embodiments include two differenttypes of supports, one for separation (e.g., superparamagneticmicrobeads or the inside surface of microtiter wells) and another fordetection and quantification (e.g., magnetically non-responsivepolystyrene microspheres of selected diameters that can be identifiedand counted in a particle size distribution analysis system such as aCoulter counter). For example, the first complexing agent on the firsthybridization probe is contacted with the second complexing agent boundto a first bead that is responsive to a magnetic field (M) eitherbefore, during, or after the first and/or second hybridization probe ishybridized to the sample of nucleic acids. By contacting the first andsecond complexing agents, the first hybridization probe becomesimmobilized on the magnetically responsive bead. This enables theimmobilization of any nucleic acid hybridized to the first hybridizationprobe, i.e., a nucleic acid that includes a nucleic acid sequence of thefirst type. The magnetically responsive bead enables nucleic acidshybridized to the first hybridization probe to be separated from nucleicacids that do not hybridize to the first hybridization probe.

Similarly, the third complexing agent on the second hybridization probeis contacted with the fourth complexing agent bound to a second bead,which is non-responsive to a magnetic field (NM) but may be of differentsize than the first bead (and/or responsive to an electric field),either before, during, or after the first and/or second hybridizationprobe is hybridized to the sample of nucleic acids. By contacting thethird and fourth complexing agents, the second hybridization probebecomes immobilized on the non-magnetic responsive bead. This enablesthe immobilization of any nucleic acid sequence hybridized to the secondhybridization probe, i.e., a nucleic acid sequence that includes anucleic acid sequence of the second type. The magneticallynon-responsive bead can then be used as a detectable marker followingmagnetic separation for those target nucleic acids that have both type 1and type 2 sequences on the same contiguous molecule. If themagnetically non-responsive bead is responsive to an electric field(e.g., by coating with carboxylic acid) and uniquely complexable to asolid support, two additional separation steps would be possible. Forexample, Step 1 could be by magnetic separation, Step 2 byelectrophoretic separation, and Step 3 by complexing the non-magneticbeads to a solid support such as a glass slide and detection byfluorescence scanning, or to a solid support such as the inside surfaceof a well in a 96 well plate. Also, step 2 separation can beaccomplished by filtration if different size beads are used, or byparticle size characterization if a particle size measurement device isemployed (these methods are taught in Example 2).

Only nucleic acids containing the first nucleic acid sequence type,i.e., nucleic acids that hybridize to the first hybridization probe,will be immobilized onto the magnetically responsive bead. Of thesenucleic acids, only those containing the second nucleic acid sequencetype will hybridize to the second hybridization probe, which isimmobilized onto the magnetically non responsive bead. Thus, after thefirst separation step by response to magnetic force, followed bywashing, the remaining target nucleic acids contain sequences hybridizedto the first probe and sequences hybridized to both the first probe andthe second probe. All target nucleic acid sequences not hybridized tothe first probe, or not hybridized to the same contiguous nucleic acidmolecule as the first probe, are washed out because they are notimmobilized to the magnetic bead.

The aberrant nucleic acids, which contain sequences hybridized to thefirst probe and second probe, can be separated by exposure to anelectric field if the second bead is responsive to an electric field, orby immobilizing the second bead to a solid support if the second bead iscoated with a member of a third pair of complexing agents which iscapable of specifically complexing with the complementary member of thethird pair of complexing agents coated on the solid support. Thedetection and quantification of nucleic acids containing both type 1 andtype 2 sequences, which is directly proportional to the number ofnucleic acid aberrations present in the sample of nucleic acidsanalyzed, can be done using a variety of available methods. For example,various detectable labels can be included on the beads, on the probes,or on the target nucleic acid, such that they can be measured byfluorescence, radioactivity, luminescence, chemiluminescence,electrochemiluminescence, spectrophotometry, and the like. Coloredbeads, both fluorescent and non-fluorescent, are commercially available(e.g., Bangs Labs, Fishers, Ind.) and also can be used fordistinguishing nucleic acid types.

The method of the present invention increases by orders of magnitude thespeed of detecting nucleotide sequence aberrations, such as chromosometranslocations, over current detection methods, including FISH assays.

Since the number of type 1 plus type 2 target sequences detected in thesample of DNA analyzed would be proportional to the number of type 1plus type 2 nucleotide sequences in the cell extract (e.g., chromosomalDNA from blood lymphocytes), the method of the present invention canalso be used in the early detection and monitoring of pre-clinicaldisease progression of malignancies, such as leukemias that areassociated with specific chromosomal rearrangements, e.g., t(9;22) ofhuman chronic myelogenous leukemia. According to this embodiment of themethod, the first and second hybridization probes are designed toselectively hybridize to a first and a second nucleic acid sequencetypes, the nucleotide sequence aberration of which is associated withand/or characteristic of a disease. Only nucleic acids containing boththe first and second nucleotide sequence types, the aberration of whichis associated with and/or characteristic of a disease, will hybridize toboth the first and second hybridization probes. As a result, after thefirst separation by magnetic force followed by washing, the separationof the beads complexed to the second hybridization probe, either byelectric force, immobilization on a solid support, bead filtration, orparticle size analysis, may be used to diagnose a disease associatedwith the particular nucleic acid aberration being detected. Examples ofdiseases that may be detected include (but are not limited to) cancerssuch as leukemia, lymphoma, melanoma, prostate, and cervical cancer.

It is within the scope of the present invention that probe-beadattachments and hybridizations of probes to target nucleotide sequencescan be performed in any order, as well as simultaneously.

In another preferred embodiment of the invention, the present method canbe used for separating and rapidly quantifying objects such as proteins,cells, organelles, and the like. Instead of using hybridization probesfor binding to the target object, antibodies or other binding molecules,such as lectins, are used. It is merely required that there be at leasttwo binding sites on the target for which there is a correspondingnumber of binding molecules. For example, a protein having two epitopescan be separated from other proteins provided that an antibody forbinding each epitope is available for carrying out the separation.Example 4 teaches the separation of cells using the methods of thepresent invention.

In another preferred embodiment of the invention, beads of differentsizes can be used for carrying out separation steps. For example, if asmall magnetically non-responsive bead is coupled to an antibody thatrecognizes one epitope and a larger magnetically responsive bead iscoupled to an antibody that recognizes another epitope, then nucleicacids, proteins, cells, organelles, and the like that bear both epitopescan be separated from other objects that lack both epitopes. Adescription of this embodiment of the invention is provided in Example 2for nucleic acids, Example 4 for cells, and Example 7 for proteins.

It will be recognized by those skilled in the art that at least thefollowing differences among beads can be used for carrying outmulti-step separations: the degree of magnetic responsiveness, thedegree of charge responsiveness, selected bead size differences,selected bead color differences, and selected complexing agents on beadsand supports.

The present invention also relates to a kit for separating andquantifying nucleic acid aberrations and diagnosing disease according tothe methods of the present invention. In general, the kits of thepresent invention include beads with complexing agents and hybridizationprobes (e.g., magnetically responsive beads coated with type 1 probesand magnetically non-responsive beads coated with type 2 probes). Thekits may also include vials with reagents, suitable solid supports,instructions for using the kit, and a calibration curve (or suitableinternal control) relating the measured quantity to the frequency ofnucleic acid sequence aberrations in the target sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 2A, and 2B show exemplary methods for separating andisolating a chromosome translocation. FIG. 1 shows the isolation of DNAfrom a sample of cells, hybridization of the DNA to first and secondhybridization probes, attaching the hybridized DNA to a first bead thatis magnetically responsive and specifically complexes with the firsthybridization probe and a second bead that is magneticallynon-responsive and specifically complexes with the second hybridizationprobe, and a first separation step accomplished by subjecting themixture of bead-attached hybridized DNA to a magnetic force followed bywashing. At this stage, the beads can could be detached from the targetDNA (e.g., by DNase treatment) and directly analyzed as described inExample 6. FIG. 1B depicts a similar procedure in which the probes areattached to a non-magnetic solid support such as the inside surface of awell of a microtiter plate, permitting step 1 separation followed bybead counting or particle size distribution analysis. FIG. 2A depictsthe second separation step of DNA that contains both type 1 and type 2sequences from a sample of cells, wherein the second bead ismagnetically non-responsive but is electrically responsive, after thefirst step separation, the DNA containing both type 1 and type 2sequences is then separated by application of an electric force. FIG. 2Bshows the second separation step of DNA that contains both type 1 andtype 2 sequences from a sample of cells, wherein the second bead iscapable of specifically complexing with the second hybridization probeand a solid support, after the first separation step, the DNA containingboth type 1 and type 2 sequences is separated by complexing the secondbead to a solid support, followed by washing.

FIGS. 3A-B illustrate a pUC 19 plasmid and a 18.2 kb DNA insert usedhere as a model system to demonstrate the feasibility of the separationmethod presented in this invention.

FIG. 4 shows the sequences of the terminal ends (SEQ ID NO:1 and SEQ IDNO:2) of the DNA insert seen in FIG. 3B and the 50mer probes (SEQ IDNOS:3-10) selected to be complementary for type 1 (one of the ends) andtype 2 DNA (the other end).

FIG. 5 shows Dynal and Bangs beads observed using a light microscope at1000× magnification. In this case, beads were taken from the stocksolutions, mixed, placed on a glass microscope slide, and viewed underoil immersion.

FIG. 6 shows a microscope image of beads deposited on a glass slideafter the bead solution (Dynal+Bangs) had been subjected to magneticseparation, but without hybridization to the 18.2 kb DNA. Note that onlyDynal beads are seen (the few small dots are dust on lens as they canalso be seen on the other images).

FIG. 7 shows a microscope image following hybridization of beads to the18.2 kb target DNA and magnetic separation. In this case, 1 μl ofSolution B was deposited on a slide and HA on the Bangs beads complexedwith the anti-HA on the surface of the slide. After washing, only theBangs beads attached to the slide. The Dynal beads, which were connectedto the Bangs beads because they were both hybridized to the same DNAmolecule, are also present on the slide. The presence of both Dynal andBangs beads on this slide demonstrates that both were hybridized to thetarget DNA and that the separation procedure was successful.

FIG. 8 illustrates the particle size distribution obtained for the twobead types following magnetic separation of the 18.2 kb target DNAsequence. In contrast with the results in Table 1, in this case, thelarger non-magnetic beads were selected to place the non-magnetic beadpeak in a region of lower background counts. The materials and methodsused to obtain these results were the same as those used for Table 1with the following differences: FIG. 8 used 4.4 μm diameter magneticallynon-responsive polystyrene beads coated with streptavidin (Bangs Labs,Fisher, Ind.) whereas Table 1 used 0.94 μm diameter magneticallynon-responsive polystyrene beads coated with streptavidin (Bangs Labs);FIG. 8 used 5 μg of 18.2 kb target DNA whereas Table 1 used 20 μg of18.2 kb target DNA; partial magnetic separation was done for FIG. 8following DNase treatment to reduce (but not fully eliminate) the numberof superparamagnetic beads to provide a lower background level while atthe same time provide a 2.8 μm peak for comparison/illustrationpurposes; and for FIG. 8 the final bead concentration was dilutedtwo-fold just before generating the bead size distributions using theCoulter Multisizer II. It is clear that separation and quantification ofthese bead types can be accomplished using our methods and commerciallyavailable particle analyzers.

FIG. 9 illustrates Type 1 and Type 2 DNA and the beads with ECL labelsand probes for detection of the non-magnetic beads byelectrochemiluminescence (ECL). Note that the hybridization could occurin any order, prior to, during, or after complexing the probes with thebeads. If attachment of probes to the beads is performed first inseparate solutions, then both bead types can use the same complexingagents (e.g., avidin-biotin). However, if the probes are hybridizedfirst (which is the preferred method) then the complexing agents bindingthe beads to the respective probes would be unique and specific for eachbead-probe complex. In this case, we could use streptavidin coatedmagnetic beads that would complex with biotinylated probes andantidigoxigenin coated non-magnetic beads that would complex withdigoxigenin labeled probes. As depicted, a magnet may be used to holdthe complex in place during measurement.

FIG. 10 shows the separation of erythrocytes containing the glycophorinMN surface proteins from all other erythrocytes.

FIG. 11 illustrates a method for evaluating for the presence, absence,or amplification of nucleic acid sequences in a sample of nucleic acid.The examples of complexing agents are as follows: 1=biotin;2=digoxigenin; 3=estradiol; 4=fluoresceine; 5=anti-digoxigenin;6=anti-estradiol; 7=anti-fluoresceine; and 8=avidin.

FIGS. 12A-D illustrate particle size spectra obtained using the particlecounting assay to detect bcr/abl fusions in genomic DNA isolated fromhuman CML cells. FIGS. 12A-C are the spectra of microparticles observedin 500 μl samples with 165 ng, 16.5 ng, and 0 ng of genomic K-562 DNA,respectively. FIG. 12D is the result obtained with 16.5 ng genomic K-562DNA when the probes were not present during hybridization.

FIG. 13 illustrates a method to simultaneously separate and quantifyselected target molecules (e.g., proteins) using antibodies and beads ofdifferent diameters. Magnetic (M) beads and non-magnetic (N) beads arecomplexed with selected antibodies that permit their unique attachmentto different antigenic sites on the same target molecule. Note that theN-beads will remain after magnetic separation only if both antigenicsites are present on the same contiguous molecule.

FIG. 14 shows the particle size distribution obtained using thedescribed method to separate ferritin.

FIG. 15 shows the results obtained by repeating the separation in FIG.14, but without ferritin in the target solution.

DETAILED DESCRIPTION OF THE INVENTION

Before the present compositions and methods for separation andquantization of aberrant nucleic acid sequences, other molecules, cells,and the like are disclosed and described, it is to be understood thatthis invention is not limited to the particular configurations, processsteps, and materials disclosed herein as such configurations, processsteps, and materials may vary somewhat. It is also to be understood thatthe terminology employed herein is used for the purpose of describingparticular embodiments only and is not intended to be limiting since thescope of the present invention will be limited only by the appendedclaims and equivalents thereof.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outherein.

In one illustrative embodiment, the present invention relates to amulti-step method for detecting and separating nucleic acid sequenceaberrations. As used herein, the term “nucleotide sequence aberration”refers to rearrangements between and within nucleotide sequences,particularly chromosomes. Nucleotide sequence aberration also refers tothe deletion of a nucleic acid sequence, particularly chromosomedeletions. As used herein, the term “nucleic acids” refers to DNA andRNA of any origin, and any level of organization, e.g., DNA molecule,chromatin, chromosome.

According to the method of the present invention, a nucleotide sequenceaberration is detected by isolating and quantifying nucleotide sequenceshaving both a first nucleotide sequence type (e.g., from a firstchromosome) and a second nucleotide sequence type (e.g., from a secondchromosome). The presence of the first and the second nucleotidesequence types on the same nucleic acid indicating the presence of anucleotide sequence aberration.

The method is referred to as a multi-step method because it involves atleast two sequential separation steps. According to the method of thepresent invention, as illustrated in FIG. 1, the nucleic acid samplecomprises chromosomal DNA isolated from a sample of cells. ChromosomalDNA may be isolated by any of the variety of methods known in the art.For example, the DNA may be isolated by the method taught in U.S. Pat.No. 5,447,841 and references therein; in Vooijs, et al., Am. J. Hum.Genet. 52:586-597 (1993); or by using the GIBCO BRL TRIzol™ Reagent(Life Technologies, Gaithersburg, Md.), or by using a QIAprep kit(Qiagen, Inc., Valencia, Calif.).

Chromosomal DNA may be analyzed as whole chromosomes, chromosomefragments, chromatin fragments, or chromosomal DNA fragments, all ofwhich are hereinafter referred to as chromosomal DNA. When analyzingchromosomal DNA for the presence of nucleotide sequence aberrations, thechromosomal DNA may be organized as an extended double strand, asextended nucleosomes, as chromatin fiber, as folded fiber, and asinterphase, prophase, or metaphase DNA. Sandberg, “The chromosomes inhuman cancer and leukemia”, Elsevier; N.Y. (1980), pp. 69-73.

The preferred chromosome organization for assaying chromosomal DNA forthe presence of a nucleotide sequence aberration depends on the numberof bases separating the first and second nucleotide sequence types beingrecognized by the first and second hybridization probes used to identifythe aberration. The preferred size of beads is a function of the size ofthe piece of target DNA or RNA to be evaluated. For example, targetpieces of DNA can range from less than a micrometer to severalmillimeters in length depending on the level of organization used andthe degree to which the chromosomes are fractionated. For example,accurate quantification of the frequency of t(9;22) fusions in chronicmyelogenous leukemia (CML) patients would require target DNA pieces onthe order of a few hundred kilobases (less than 1 mm) if the DNAmolecules are fully extended and only a few micrometers if thechromosomes are in the interphase level of organization. (Note that theposition of the fusion point in CML patients can vary by about 225 kb.)

Although it is within the scope of the present invention that probe-beadattachments and hybridizations of probes to target nucleotide sequencescan be performed in any order, as well as simultaneously, as illustratedin FIG. 1, it is a preferred embodiment of this invention that thefollowing order be used to reduce the number of complexing agentsrequired (e.g., see Example 1). Two different probe-bead complexes areprepared in separate solutions. Solution I would contain the firsthybridization probe with a member of a pair of first complexing agentscapable of attaching to a complementary member of the first pair ofcomplexing agents. Also contained in Solution I would be magneticallyresponsive beads coated with the complementary member of the pair offirst complexing agents. The beads and hybridization probes would becombined in Solution I such that complexing of the pair of firstcomplexing agents takes place. Subsequent washing would remove allprobes and reagents not complexed with the beads. Solution II wouldcontain the second hybridization probe with a member of a second pair ofcomplexing agents capable of attaching to a complementary member of thesecond pair of complexing agents. Also contained in Solution II would bemagnetically non-responsive beads coated with the complementary memberof the second pair of complexing agents. The beads and hybridizationprobes would be combined in Solution II so as to permit complexing ofthe second pair of complexing agents. Subsequent washing would removeall probes and reagents not complexed with the beads. The magneticallynon-responsive beads in Solution II may also be coated with a member ofa third pair of complexing agent to facilitate a particular Step 2separation embodiment, if desired (see below).

The two types of probe-bead complexes are then combined in one solutionand contacted with a sample of target nucleic acid sequences underconditions favorable for hybridization. The first hybridization probeincludes a nucleotide sequence probe that is at least partiallycomplementary to a first target nucleotide sequence type. The secondhybridization probe includes a nucleotide sequence that is at leastpartially complementary to a second nucleotide sequence type and thatselectively hybridizes to the second nucleotide sequence type over thefirst nucleotide sequence type.

In the case of detecting and separating chromosomal translocations, thefirst hybridization probe is preferably a chromosome-specific probe suchthat it selectivity hybridizes to a particular chromosome type. In thecase of detecting inter-chromosomal rearrangements, “chromosome type”refers to individual chromosomes. In the case of detectingintrachromosomal rearrangements, “chromosome type” refers to differentportions of an individual chromosome since intra-chromosomalrearrangements involve the movement of a sequence to a different portionof the same chromosome.

Any hybridization probe that preferentially hybridizes to a particularnucleotide sequence may be used as the first hybridization probe and isintended to fall within the scope of the present invention. In the caseof detecting chromosome translocations, a large number of bothchromosome-specific painting probes and unique sequence probes areavailable commercially (e.g., Oncor, Inc., Gaithersburg, Md.; or Vysis,Inc. (www.vysis.com)). In addition, methods are broadly available foranyone skilled in the art to prepare nucleic acid probes that arecomplementary to any known sequence. For example, thousands of humangenes have now been mapped to specific regions of chromosomes andsequenced. These sequences are now available on the Internet and can beused to make biotinylated type 1 and type 2 probes for use in thepresent invention to quantify DNA rearrangements (see Example 1).Exemplary methods for preparing DNA probes for chromosome translocationdetection are given in U.S. Pat. No. 5,447,841, which is herebyincorporated herein by reference. A preferred modification to the methodof U.S. Pat. No. 5,447,841 is the use of biotin TEG phosphoramidite toattach individual biotin molecules to the 5′ end of the probes (seeExample 1).

Because the first hybridization probe is selective for a firstnucleotide sequence type as opposed to the nucleotide sequenceaberration itself, the first hybridization probe hybridizes to allnucleotide sequences containing the first nucleotide sequence type. Forexample, with regard to detecting a chromosome translocation, the firsthybridization probe may be a chromosome-specific probe. Thus, the firsthybridization probe does not by itself detect the nucleotide sequenceaberration. Rather, the method of the present invention relies on thesecond hybridization probe to identify those nucleotide sequencesisolated by the first hybridization probe that also have a nucleotidesequence of a second type. By contrast, in most prior art hybridizationassays using two hybridization probes, the first hybridization probeselectively isolates the nucleic acid being detected while the secondhybridization probe serves to enable detection of the nucleotidesequence isolated by the first hybridization probe.

The first hybridization probe also includes a complexing agent that isconfigured for binding to a complementary complexing agent for forming afirst pair of complexing agents. The complementary complexing agent isattached to a first bead, which is responsive to magnetic force, therebyenabling the immobilization of the first hybridization probe on themagnetically responsive bead. The second hybridization probe includesanother complexing agent that is configured for binding to anothercomplexing agent for forming a second pair of complexing agents. Thecomplementary complexing agent of the second pair of complexing agentsis attached to a second bead, which is non-responsive to magnetic force,but is either electrically responsive, is coated with a complexing agentthat is configured for binding to still another complementary complexingagent for forming a third pair of complexing agents, and/or is ofdifferent size than the first bead. The complementary complexing agentof the third pair of complexing agents is attached to a solid support,thereby enabling the immobilization of the second hybridization probe onthe solid support.

The first, second, and third pairs of complexing agents may be any pairof complexing agents that form a strong binding pair. Since elevatedtemperatures may be required for hybridization, the binding pair shouldpreferably be stable at temperatures at least up to about 40° C.

Examples of suitable binding pairs of complexing agents includebiotin-avidin, and antibody-antigen pairs, such as hemagglutinin andanti-hemagglutinin, and digoxigenin and anti-digoxigenin. Avidin-biotinand analogues and derivatives thereof are particularly preferred asbinding pairs due to their enhanced thermal stability.

Magnetically responsive and magnetically non-responsive beads suitablefor the present invention are commercially available (Dynal AS, Oslo,Norway; Bangs Labs, Fishers, Ind.). Preferably the first bead ismagnetically responsive and the second bead is non-responsive tomagnetic force, but is either responsive to electric force, coated withan additional complexing agent (e.g., a peptide for step 2 antibodyseparation), or smaller in size than the first bead, thus facilitatingstep 2 separation by filtration or by particle size distributionanalysis (see Example 2). Preferably, both bead types are coated withavidin as a complexing agent for attachment to uni-labeled biotinylatedprobes (i.e., only one biotin per probe). The magneticallynon-responsive beads may also be coated with, e.g., carboxylic acid,which provides a negative surface charge and which would make themresponsive to electric force.

Any solid support to which a complexing agent may be attached may beused in the present invention. Examples of suitable solid supportmaterials include, but are not limited to, silicates such as glass,plastics, polyethylene, cellulose and nitrocellulose, polymethacrylate,latex, rubber, fluorocarbon resins such as TEFLON, metals, nylon,polystyrene, and the like.

The solid support material may be used in a wide variety of shapesincluding, but not limited to microscope slides, microspheres, andmicrotiter wells. Examples are provided herein of attachments to glassmicroscope slides and quantification by fluorescence scanning ormicroscopy (Example 1), and also of the use of different bead sizes,filtering, and quantification by bead counting or size characterization(Example 2).

Preferably, avidin or an avidin derivative is used in both the first andsecond pairs of complexing agents. Magnetically responsive andmagnetically non-responsive microbeads labeled with streptavidin may beobtained from Dynal AS, Oslo, Norway, or Bangs Labs, Fishers, Ind. (seeExample 1).

The first and second hybridization probes may be immobilized to thefirst bead or second bead either before, during, or after the first andsecond hybridization probes are hybridized to the sample of targetnucleic acids. The first and second hybridization probes are preferablyattached to the beads in separate solutions before the probes arehybridized to the sample of nucleic acids. This permits the use ofbiotin-avidin complexing agents for both bead types. Note, in this case,only one biotin is attached to each probe such that after binding to thebeads and washing there is no unbound biotin on the probes to permitcross reaction between the beads (see Example 1).

Once target nucleic acids have been hybridized to the first and secondhybridization probes immobilized on the first and second beads, thenucleic acids that hybridized with the first hybridization probe may beisolated by subjecting the hybridization mixture to a magnetic forcefollowed by washing. Any non-hybridized nucleic acids and nucleic acidsnot containing type 1 sequence are washed out because they are notcomplexed with the magnetically responsive bead.

The second hybridization probe includes a nucleotide sequence that doesnot hybridize to nucleic acids of the same type as the firsthybridization probe. Any nucleotide sequence that does not hybridize tothe first target nucleotide sequence type may be used in the secondhybridization probe and is intended to fall within the scope of thepresent invention. Both hybridization probes may include analyticallydetectable markers that could be used to quantify the frequency of thenucleotide sequence aberration being detected. Furthermore, both typesof beads can be tagged with detectable markers using methods availableto someone with ordinary skill in the art. Also, beads are now availablecommercially in a wide selection of colors, e.g., from Bangs Labs,Fishers, Ind.

Optionally, the second hybridization probe may hybridize to more thanone chromosome type other than the chromosome type to which the firsthybridization probe hybridizes, e.g., a heterogeneous mixture of uniquesequences selected from many non-type 1 chromosomes (see U.S. Pat. No.5,447,841). In this embodiment the second hybridization probe wouldpermit the detection of a larger fraction of the nucleotide sequenceaberrations involving the chromosome identified by the firsthybridization probe.

Where possible, the hybridization probe preferably includes a nucleotidesequence that is uniquely specific to the nucleotide sequence aberrationbeing detected. The use of uniquely specific hybridization sequences ispreferred since it minimizes the occurrence of background noise due tononspecific hybridization. It is also possible to use a composite secondhybridization probe that includes a series of sequences that are alleither unique or chromosome specific for the aberration being detected.Such a composite “cocktail” of probes, if each with detectable markers,would enhance the signal to be detected and thus potentially decreasethe limit of detection for the assay.

After the first step of magnetic force separation, the remaining targetnucleic acids contain type 1 sequence complexed with the magneticresponsive beads, type 1+non complexed with magnetic responsive beads,or type 1+type 2 sequences if a rearrangement (fusion) between type 1and type 2 nucleic acids has occurred (see FIG. 1). FIGS. 2A and 2Billustrate two embodiments of the second separation step of the presentinvention. According to FIG. 2A, the second bead used is non-responsiveto magnetic force, but responsive to electric force. After hybridizationand the first step of magnetic force separation described above, thenucleic acids that contain both type 1 and type 2 sequences can beseparated by application of an electric force, because only nucleicacids containing both type 1 and type 2 can be complexed with the secondbead, which is electrically responsive. It should be noted that sincethe nucleic acids containing both type 1 and type 2 sequences areresponsive to both magnetic and electric forces, they can be purifiedthrough additional cycles of application of magnetic and electricforces.

According to FIG. 2B, the second bead used is non-responsive to magneticforce, but is coated with another complexing agent in addition tocomplexing agent from the second pair of complexing agents. Thisadditional complexing agent is configured for forming a specific complexwith a complementary complexing agent that is coated on a solid support.The combination of these two complexing agents forms a third pair ofcomplexing agents. After hybridization and the first step of magneticforce separation described above, the nucleic acids containing both type1 and type 2 sequences can be separated by immobilization on the solidsupport, because only nucleic acids containing both type 1 and type 2can be complexed with the solid support by forming a complex between thethird pair of complexing agents. After washing to remove unattachedbead-DNA complexes, the immobilized nucleic acids containing type 1 andtype 2 sequences may be detected by a variety of methods known in theart including, but not limited to, automated fluorescence scanning(e.g., tagged beads in a miniarray format), microscopy, etc. (seeExample 1).

According to the method of the present invention, the first separationstep enables the separation of nucleotide sequences of a firstnucleotide sequence type. Since the second hybridization probe isdesigned so that it does not hybridize to nucleotide sequences of thefirst nucleic acid type, the second hybridization probe does not bind tonucleic acids immobilized by the first hybridization probe that do notcontain a nucleotide sequence aberration. As a result, the number oftarget nucleic acid fragments (or metaphase chromosomes) that containboth type 1 and type 2 sequences isolated after the second step isproportional to the number of nucleotide sequence aberrations in thesample of nucleic acids being analyzed.

Because the magnetic separation step resulted in the elimination of allnucleic acids that did not contain at least some type 1 DNA, the resultof the second separation step would be to end up with only nucleic acidsthat contain both type 1 and type 2 DNA. The second step could also beaccomplished using other separation detection methods, such as bead sizecharacterization, or filtration and bead counting, taught in Example 2.Also, if the second step was electrophoresis separation, a third stepcould be added for separation and quantification such as depositing theelectrophoretically-separated bead complexes onto a miniarray on a glassslide and detection of fluorescence labels (e.g., Cy-3) on the beads byautomated fluorescence scanning. This is a sensitive and efficientmethod that, theoretically, can detect as little as one bead per arrayspot.

Another format claimed in the present invention which facilitatesautomated processing and detection of nucleic acid aberrations is 96well (or other format) plates.

If non-unique “painting probes” are used, nonspecific binding by thenon-unique hybridization probes to the nucleic acid sample may beminimized through the use of non-specific sequence blocking techniquessuch as those disclosed by U.S. Pat. No. 5,447,841, and Pinkel et al.,85 Proc. Natl. Acad. Sci. USA 9138-9142 (1988), which are herebyincorporated herein by reference.

The first and second hybridization probes may include RNA or DNAsequences such that the complementary nucleotide sequences formedbetween the hybridization probes and the target sequence may be two DNAsequences or an RNA and a DNA sequence.

The detection and quantification of isolated sequences containing type1+2 sequences can be done using a variety of available methods, such astotal DNA measured by spectrophotometry, various labels on the beads orprobes such that they can be measured by chromatography, fluorescence,isotopes, and the like. Any analytically detectable label that can beattached to or incorporated into a hybridization probe or bead may beused in the present invention. An analytically detectable label refersto any molecule, moiety, or atom that can be analytically detected andquantified. Methods for detecting analytically detectable labelsinclude, but are not limited to, radioactivity, fluorescence,absorbance, mass spectroscopy, EPR, NMR, XRF, luminescence, andphosphorescence. For example, any radiolabel that provides an adequatesignal and a sufficient half-life may be used as a detectable label.

Fluorescent molecules, such as fluorescein and its derivatives,rhodamine and its derivatives, cyanide and its derivatives, dansyl,umbelliferone and acridimium, and chemiluminescent molecules such asluciferin and 2,3-dihydrophthalazinediones may also be used asdetectable labels. As discussed herein, the nucleotide sequences used inhybridization probes may themselves function as detectable labels wherethe bases forming the nucleotide sequence are quantified usingtechniques known in the art.

Also, beads can be detected by directly counting as in Example 2 usingdifferent bead sizes, or by fluorescence intensity emitted fromminispots, on a glass slide detected by automated fluorescence scanning.A large number of fluorescent tags and methods for attaching to nucleicacid probes and beads are now available commercially.

A nucleotide sequence aberration frequency (e.g., number of aberrantsequences per total number of cells from which the DNA sample wasobtained) may be determined based on the signal generated from thedetectable marker using a calibration curve. The calibration curve maybe formed by analyzing a sample of cells having a known nucleotidesequence aberration frequency. For example, the FISH method fordetecting chromosome translocations may be used to determine thenucleotide sequence aberration frequency rate of a sample of cells.Then, by serially diluting the sample of cells and assaying the cellsaccording to the method of the present invention, a calibration curvemay be generated. Alternative methods for generating a calibration curveare within the level of skill in the art and may be used in conjunctionwith the method of the present invention.

Interchromosomal rearrangements typically are of two types:translocations and dicentrics. Translocation are rearrangements thatresult in two derivative chromosomes that have one centromere each,whereas dicentrics are rearrangements that result in one derivativechromosome with two centromeres and another with no centromeres. Whenquantifying the frequency of interchromosomal rearrangements, it isoften useful to know whether they are translocations or dicentrics.Translocations persist for a lifetime, while dicentrics diminish withtime. Dicentric chromosomes may be identified according to the method ofthe present invention by using first and second hybridization probesthat each hybridize to the centromere of a different chromosome. DNAprobes specific to the centromeres for almost all human chromosomes arenow commercially available for (e.g., Ventana Medical Systems, Inc.,Tucson, Ariz.; Oncor, Inc., Gaithersburg, Md.).

The present invention also relates to a kit for separating andquantifying nucleic acid aberrations and diagnosing diseases accordingto the methods of the present invention. In general, the kits of thepresent invention include a first hybridization probe and a secondhybridization probe as described herein. The kits may also include acomplexing agent bound to a magnetic responsive bead, one or morecomplexing agents bound to a bead that is magnetically nonresponsive.The kit may also be designed for a 96-well plate format, either with orwithout magnetic beads, as described herein as well as instructions forusing the kit. The kits may also include beads of different sizes,colors, and detectable markers.

All publications, patents, patent applications, and commercial materialscited herein are hereby incorporated by reference.

EXAMPLES

The following examples are given to illustrate various embodiments whichhave been made within the scope of the present invention. It is to beunderstood that the following examples are neither comprehensive norexhaustive of the many types of embodiments which can be prepared inaccordance with the present invention.

Example 1

This is an example of a method that can be used to separate and quantifyDNA with two unique and non-overlapping sequences. In this example, thesequences (identified here as Type 1 and Type 2 DNA) span 0.8 kb eachand are about 17 kb apart on a contiguous 18.2 kb double stranded DNAmolecule. Importantly, these sequences are too far apart for detectionby PCR, which is limited to less than about 10 kb, and the 18.2 kbextended DNA molecule is too small for detection by FISH which isgenerally limited to the detection of condensed DNA such as that inmetaphase chromosomes. Hence, the separation and detection demonstratedin the present example could not have been accomplished using availablemethods. It is also important to note that the DNA molecule selected forthis example could just as well have been the result of a rearrangementbetween Type 1 and Type 2 DNAs, where, for example, Type 1 DNA is fromone chromosome and Type 2 DNA is from another chromosome. Hence, themethod of the present example can be used to separate and quantify anyrearrangement for which complementary Type 1 and Type 2 DNA probes areavailable or obtainable. A large number of probes are now available forsequences that flank DNA fusion points associated with cancer-relatedchromosomal rearrangements, such as the t(9;22) observed in humanmyelogenous leukemia and described in Tkachuk et al., 250 Science559-562 (1990), and U.S. Pat. No. 5,487,970.

It is also possible to use the method of the present example to separateand quantify random rearrangements that may be induced by clastogenicagents such as radiation and certain chemicals. In this case, the objectwould be to sample as large a fraction of the genome as possible byselecting the largest chromosomes as hybridization targets. For example,if Type 1 DNA probes were selected to be unique to chromosome 1 (e.g., asequence, or cocktail of sequences, complementary to one or more geneslocated on chromosome 1, or a human chromosome 1 centromeric probeavailable from Oncor, Inc., Gaithersburg, Md.), and Type 2 DNA probeswere selected to be unique to chromosomes other than chromosome 1 (e.g.,a cocktail of composite probes complementary to gene sequences onchromosomes 2 through 4), any chromosome 1 with Type 2 DNA probesattached would be the result of a translocation between chromosome 1 andchromosomes 2, 3, and/or 4. The present method would quickly separateand quantify such events. A large number of chromosome-mapped genesequences are now available from human genome projects. In fact, achromosome-specific physical map now exists for over 30,000 human genes(Deloukas et al., Science 282, 744-746; 1998) and a large number ofthese genes have sequences published on the Internet (e.g.,www.ncbi.nlm.nih.gov). Importantly, anyone skilled in the art can useavailable methods (described below) and these published sequences tosynthesize biotinylated probes that are complementary to the availablegene sequences and thus make biotinylated probes (or cocktails ofcomposite biotinylated probes) that are unique to any human chromosome,even unique to many regions of human chromosomes. The probes could alsobe painting probes (e.g., commercially available from Vysis, Inc., orVentana Medical Systems, Inc. (Tucson, Ariz.). For painting probes,non-specific hybridization can be reduced using available non-specifichybridization blocking methods (Pinkel et al., Proc. Natl. Acad. Sci.USA 9138-9142, 1988; U.S. Pat. No. 5,447,841). By usingchromosome-specific (unique) probes (e.g., complementary to gene codingsequences) for hybridization to Type 1 DNA, the first separation stepwould be assured to be very clean. For the special case of separationand quantification of dicentric chromosomes (i.e., interchromosomalrearrangements resulting in a chromosome with two centromeres),centromeric probes can be used for both Type 1 and Type 2 DNA. Theseprobes are available commercially for essentially all human chromosomes(e.g., Oncor, Inc).

Target DNA

The DNA used as hybridization target for this example of the presentmethod is an 18.2 kb insert in a pUC19 plasmid (FIGS. 3A-B). Thedouble-stranded DNA insert was positioned at the plasmid's SalI site sothat it could be removed from the plasmid by digestion with SalIrestriction enzyme.

The DNA insert was amplified by growing the plasmid in E. coli bacteriaas follows. First, 4.2 μg of plasmid DNA was diluted in 1000 μldistilled deionized water. One μl of this solution was then used toelectroporate the DNA into E. coli using a Bio-Rad electroporationapparatus (Bio-Rad, Inc.). The E. coli were immediately collected andplaced in an incubator at 37 C for 30 min, followed by plating on agarmedium (Luria-Bertani with ampicillin), and incubating at 37 Covernight. The next day, individual colonies were collected and grownovernight in 5 mL of liquid medium (Luria-Bertani with ampicillin).

The plasmid DNAs were purified using a QIAprep-spin plasmid kit (#27104)according to manufacturer's protocol in QIAprep Miniprep Handbook, April1998, pp. 18-19 (Qiagen, Inc., 28159 Stanford Ave., Valencia, Calif.91355).

The purified plasmid DNAs were then digested with SalI to permitextraction of the 18.2 kb target DNA insert. The Sal I restrictionenzyme was obtained from Sigma at a stock concentration of 10,000units/mL. To the plasmid solution (30 μg/132 μl TE) was added 15 μl SalIstock solution, 30 μl Sal I buffer from Sigma, and 135 μl distilleddeionized water. After mixing, the reaction mixture was incubated at 37C for 1 hour, then maintained at 4° C. overnight. Before use, solutionswere changed to fresh aliquots of B&W buffer (10 mM Tris-HCl, pH 7, 1 mMEDTA, 2 M NaCl).

Then, the 18.2 kb target DNAs were extracted usingphenol/chloroform/isoamyl alcohol (25:24:1; v:v:v) as described in“Molecular Cloning: A Laboratory Manual”, Second Edition, Sambrook etal., Eds. (1989) and washed with ether. DNAs were precipitated with 100%ethanol after adding 3 M NaOAc to final concentration 0.3 M. Theprecipitated solutions were then stored at 0 C for 2 hours, centrifuged,and the supernates removed. Residues were washed with 70% ethanol anddried at room temperature in air.

The extracted 18.2 kb DNAs were verified by agarose gel electrophoresisusing standard methods.

DNA Probes

For the present example, the DNA sequences (50mers) used ashybridization probes were synthesized using an Applied Biosystems Model3948 synthesizer (Perkin-Elmer, Applied Biosystems Division, FosterCity, Calif.) and standard methods known to those skilled in the art ofDNA oligonucleotide synthesis to be uniquely homologous with theterminal ends of the 18.2 kb insert. First, about 1 kb of DNA wassequenced at each terminal end of the 18.2 kb insert. Then, four 50 bpcomplementary sequences, each with uniquely different sequences, weresynthesized with homology to one end of the DNA insert (Type 1 Probes)and another four 50 bp complementary sequences synthesized with uniquehomology to the other end of the DNA insert (Type 2 Probes).

The probes were then biotinylated by covalently attaching biotin TEGphosphoramidite (Cat. #10-1955-02, Glen Research, Sterling, Va.) to the5′ end of the DNA probes via a 15 atomic bond spacer arm. The result isthat no probe has more than one biotin attached to it, and hence thereis no unbound biotin on the probe after complexing with avidin on thesolid support and washing. This is useful in the present inventionbecause all probe types (as well as any other attachments to the beadssuch as probes for detection and peptides for other unique complexingagents) can use biotin as complexing agents to attach them to theavidin-coated beads as long as the attachments to the beads of differentprobe types are performed in separate solutions. Using a spacer armgreater than 10 to 12 atomic bonds eliminates potential binding problemsbetween the biotinylated probes and the beads, and also facilitatessubsequent hybridization of the probe to the target DNA (A. J. Ninfa andD. P. Ballou, “Fundamental laboratory approaches for biochemistry andbiotechnology”, pp. 102-103, 1998, Fitzgerald Science Press, Inc.,Bethesda, Md.).

The sequences of the terminal ends of the 18.2 kb DNA and the unique50mer probes are shown in FIG. 4. Sequencing of the ends of the 18.2 kbDNA was performed using ABI Prism BigDye Terminators and cyclesequencing with Taq FS DNA Polymerase. DNA sequences were collected andanalyzed on an ABI Prism 377 automated DNA sequencer (Perkin-Elmer,Applied Biosystems Division, Foster City, Calif.).

Beads

Two types of beads were used in this example, magnetically-responsivebeads purchased from Dynal AS, Oslo, Norway, and magneticallynon-responsive beads purchased from Bangs Labs, Fishers, Ind.

The magnetically responsive beads were “Dynabeads Streptavidin” (Product#112.05), which are 2.8 μm in diameter and coated with streptavidin. Themagnetically non-responsive beads were from Bangs Labs (Catalog #CP0IN), which are 0.94 μm in diameter and also coated with streptavidin.The two bead types are seen in FIG. 5 (1000× magnification, lightmicroscope).

The two bead sizes selected provided a simple and very useful means toverify results as the technology was being developed. That is,hybridization and separation efficiencies are rapidly determined byobserving bead types at various stages using a light microscope.

Attach Probes to Beads

The biotinylated probes were then attached to the Dynal avidinylatedbeads as follows. First, 0.2 mL TE buffer solution (10 mM Tris-HCl, pH7.5, 1 mM EDTA) containing 2.8 μg Probe 1, 2.8 μg Probe 2, 2.8 μg Probe3, 2.8 μg Probe 4, and 1.3 μg of a biotinylated T7 22mer (Cat. #300322,Stratagene.com) was prepared. Probes 1 through 4 were the 50mers thatwere synthesized to be complementary to the DNA on one end of the 18.2kb target DNA (Type 1 DNA). The T7 22mer is a unique probe used here fordetection purposes only, i.e., it provides an option for subsequenthybridizations with a fluorescent marker to detect the presence of theDynal beads.

Next, the Dynal beads (2 mg/0.2 mL) were washed once with 1 mL of B&Wbuffer solution and resuspend in 0.2 mL B&W buffer. For themagnetically-responsive Dynal beads, washing was accomplished using theMagnetic Particle Concentrator (MPC) (Dynal AS).

Finally, the solution of Probes 1 through 4 and T7 was mixed with thesolution of Dynal beads. This mixture was then gently shaken for 1 hourat room temperature. The beads were then washed four times with B&Wbuffer, 0.4 mL each. Next, the probe-coated Dynal beads were resuspendedin 0.4 mL B&W and stored at 4 C.

The biotinylated probes were attached to the Bangs avidinylated beads asfollows. Similar to the preparation of the Dynal beads described above,0.2 mL TE buffer solution was prepared containing 2.8 μg Probe I, 2.8 μgProbe II, 2.8 μg Probe III, 2.8 μg Probe IV, and 12 μg of a biotinylatedhemagglutinin (HA) peptide. Probes I through IV were the 50mers thatwere synthesized to be complementary to the DNA on the other end of the18.2 kb target DNA (Type 2 DNA). The HA peptide was synthesized using anAdvanced ChemTech Model 348 peptide synthesizer (Advanced ChemTech,Inc., www.peptide.com) and was biotinylated by attaching a single biotinmolecule to the terminal amino-end of a 6 carbon spacer molecule thatwas then attached via its carboxyl end to the terminal amino end of theHA peptide. The biotinylated HA peptide was then attached to the Bangsbeads to be used subsequently as a complexing agent for stage 2separation involving anti-HA antibody.

Next, the Bangs beads (2 mg/0.2 mL) were washed once with 1 mL of B&Wbuffer followed by one wash with 1 mL of TE. The beads were resuspendedin 0.2 mL B&W solution. For the Bangs beads (which were non-magnetic),washing was accomplished using centrifugation.

Finally, the solutions of biotinylated probes I through IV andbiotinylated HA peptide were mixed with the solution of Bangs beads.This mixture was gently shaken for 1 hour at room temperature, and thenthe reaction solution was removed by centrifugation. The beads were thenwashed once with 1:1 (v/v) B&W buffer and TE, 0.4 mL each. Theprobe-coated Bangs beads were then resuspended in 0.4 mL B&W and storedat 4 C.

Hybridization and Magnetic Separation (Usually Stage 1 Separation)

The 18.2 kb target DNA was hybridized to probes on beads. Selectedamounts (typically μg quantities) of the 18.2 kb DNA were dissolved in300 μl 70% formamide denaturing solution (210 μl formamide, 30 μl20×SSC, 60 μl distilled deionized water), then heated to 70 C for 5 min.The hot solution was immediately added to the cooled solution containing10 μl Dynal beads (2 mg/0.2 mL TE), 10 μl Bangs beads (2 mg/0.2 mL TE),75 μl 20×SSC, and 16.6 μl distilled deionized water. After mixing, 4.2μl 10% SDS and 4.2 μl salmon sperm DNA were added. Hybridization wascarried out in an incubator for 15 hours at 40 C with constant rotationof about 1 rpm. After cooling to room temperature, MPC was used toremove hybridization solution and unhybridized Bangs beads. Theremaining beads were then washed once with 600 μl 1×SSC, 0.2% SDS, for 6min at room temperature. The MPC washing step was then repeated. Thebeads were next washed with 600 μl solution of 0.1× SSC, 0.2% SDS, for10 min at room temperature. The MPC step was repeated, and then thebeads were washed three times with distilled deionized water. The beadswere next resuspended in 500 μl PBS. A 50 μl aliquot was taken assolution B (1.34×10⁴ Dynal beads/L), from which 5 μl was mixed with 45μl PBS to become solution C (1.34×10³ Dynal beads/L). The remaining 450μl solution was removed by MPC and 45 μl PBS was added to becomesolution A (1.34×10⁵ Dynal beads/1 L). Note: PBS buffer is 0.14 μgNaH₂PO₄, 0.79 μg Na₂HPO₄, 8.1 μg NaCl in total 1000 mL distilleddeionized water.

Filtration Separation

If the magnetically responsive beads in solutions A, B, and C above wereselected to be larger than the magnetically non-responsive beads, thenStage 2 separation could be accomplished by cutting the DNA connectingthe two bead types (i.e., the 18.2 kb DNA molecule hybridized to bothbead types can be digested by DNase or released from themagnetically-responsive bead via a cleavable linker) and filter thesolution through a filter selected to permit only the smallernon-magnetic beads to pass through. The number of small beads could thenbe quantified using available methods such as a Coulter counter. Also,if a cleavable linker is used to release the DNA molecule from themagnetic bead then the DNA would be pulled along with the smallnon-magnetic bead through the filter and would thus permit recovery ofthe target DNA. Importantly, the number of small beads recovered afterfiltration should be proportional to the number of target DNA molecules(i.e., Type 1+Type 2 DNA) in the hybridization solution. An example ofthe filtration method is described in Example 2 below.

Antibody-Peptide Separation

Attach antibody to glass slides. Streptavidin coated microscope slidesobtained from Cell Associates, Inc. (www.cel-1.com) were washed 3 timeswith B&W buffer solution, then immersed in 20 mL solution containing 100μg anti-HA-biotin (i.e., 10 mL TE, 9.5 mL Tris, 0.5 mL of 200 μganti-HA-biotin/mL). The slides were shaken for 30 min at roomtemperature, then allowed stand at room temperature for 18 hours. Next,the slides were washed five times (2 min each) with PBS. After airdrying at room temperature, the slides were stored at 4 C. Theanti-HA-biotin was purchased from Boehringer Mannheim Corporation(Indianapolis, Ind.). Then, 1 μl each of solutions A, B, and C (fromStage 1 separation described above) was placed on the anti-HA coatedslide at room temperature. After about 30 min, but before the 1 μl spotswere dry, the slide was transferred into a slide box with a small amountof water in the bottom of the box (the water should not come in directcontact with the slide). The slides were incubated at 4 C overnight,then washed twice with PBS and once with distilled deionized water. Theslides were then dried at room temperature.

Detection and Quantification

Light microscopy. The beads deposited on the slides were then viewedusing a Nikon microscope at 1000× total magnification (100× objectiveand 10× eyepiece). FIG. 6 shows a microscope image of a bead solution B(described above) deposited on a slide after probe attachment andmagnetic separation, but without hybridization to the 18.2 kb targetDNA. Note that no Bangs beads are observed. This demonstrated that Bangsbeads are washed away during the magnetic separation step if notconnected to the Dynal beads via hybridization to the 18.2 kb DNAmolecule.

In contrast, FIG. 7 shows a microscope image of bead solution B whichwas hybridized to the 18.2 kb DNA. In this case, the bead-probecomplexes were hybridized to the 18.2 kb DNA followed by magneticseparation as described above under “Hybridization and MagneticSeparation.” Note that many of the smaller Bangs beads were present inFIG. 7 and were pulled along during the magnetic separation step bytheir attachment via hybridization to the same DNA molecule that theDynal beads were hybridized.

Fluorescence scanning. In addition to light microscopy analysis, beadsdeposited on glass slides have also been analyzed using a fluorescencescanner. Fluorescence intensities were measured of 0.1 μl spots of beadsdeposited in an array format on a glass slide. The measurements weremade using a Molecular Dynamics fluorescence scanner (Avalanche model).Each spot contains an average of about 33 Dynal beads and was about 1 mmin diameter. After depositing on the slide, the beads were hybridizedwith a Cy-3 labeled 22mer complementary to the T7 22mer that waspreviously attached to the Dynal beads. The complementary 22mer wassynthesized using standard methods and a Cy-3 molecule was attached tothe 5′ end of the 22mers using Cy3-CE-Phosphoramidite (Glen Research,Sterling, Va.). Based on the present results, the fact that eachstreptavidin-coated Dynal bead can attach about 500,000 oligonucleotides(Dynal AS, Oslo, Norway), and the detection limit of commerciallyavailable fluorescence scanners which are on the order of 1 to 10 Cy-3per m² (Bowtell, 21 Supplement, Nature Genetics page 31, 1999), themethod described herein should be able to detect as little as a singlebead deposited on a glass slide using Cy-3 labeling and acommercially-available fluorescence scanner. Similar detection limitswould be expected for other common fluorescence labels. In practice,each bead type could be tagged in advance and thus identified at anystage of separation using a fluorescence scanner.

Particle counting. As described below in Example 2, if beads ofdifferent sizes are used with the present technology, each size coatedwith a unique probe, then the different sized beads can be selected byfiltration and counted using a particle counter (e.g., from Coulter).This is a very quick, accurate, and low-cost method to quantify thenumber of beads which, after hybridization and magnetic separation,would be proportional to the number of target DNA molecules (or othertarget objects) in the analysis solution (see Example 2 for details).

It is also possible (as described in Example 2) to eliminate thefiltration step by using a particle counter that measures both number ofparticles and their sizes (e.g., the Multisizer II by Coulter). In thiscase, distributions of particle sizes would be obtained and the numberof beads of any selected size can be quantified by integrating thedistributions (e.g., see Examples 6 and 7).

Electrophoresis Separation

It is also possible to perform separation using electrophoresis of themagnetically non-responsive beads, if such beads are selected to beelectrically charged (e.g., this would permit the use of electrophoresisfor Stage 2 separation and antibody-peptide separation for Stage 3). Anexperiment was performed in which two bead types were used,magnetically-responsive beads coated with amino groups (positive surfacecharge) and magnetically non-responsive beads coated with carboxylicacid (negative surface charge). The beads were from Bangs Labs (#MC05N,magnetic; #DC04, non-magnetic). The beads were selected to have twodifferent colors, the magnetic beads were brown and the non-magneticbeads were green. Both bead types were about 1 μm diameter.

A 2 mm hole was drilled through a 10 mL plastic pipette (the hole wasdrilled at the 5 mL mark through one side only) and the pipettesubmerged in a standard TAE electrolyte buffer (pH 8.3) of a standardelectrophoresis apparatus. The two types of beads were then mixed 1:1 inTAE buffer (pH 8.3) and 1 mL injected through the hole in the center ofthe pipette. The electrophoresis was carried out at 5 Volts per cm and aphotograph taken at the start of the electrophoresis and again at +20min. The results clearly showed that the green and brown beads weremixed at T=0 but were separated by about 1.5 cm at T=20 min (i.e., thenegatively charged non-magnetic beads moved about 0.75 mm per min). Thisdemonstrated the feasibility of using electrophoresis to separatenon-magnetic beads from magnetic beads.

Based on these electrophoresis results, the magnetic separation resultsprovided in this example, and the electrophoretic mobility measurementsof similar beads made by Ottewill et al., Kolloid Zh., 218, 34 (1967),it is apparent that a magnetic bead with essentially neutral surfacecharge should be pulled along with the electrophoretically-responsivenon-magnetic beads if the beads were connected to the same DNA moleculein a buffered solution. Magnetically-responsive beads with varioussurface charges (including essentially neutral surface charge) arecommercially available (e.g., Bangs Labs, Fishers, Ind.).

Example 2

As discussed above, Stage 2 separation and quantification can also beaccomplished by particle counting and size distribution analysis.

Filtration. For example, if Stage 1 separation is by magnetic force andStage 2 is by filtration of beads, then the magnetically responsivebeads would simply be selected to be larger than the non-magnetic beads.This would permit rapid separation of magnetically-responsive beads fromnon-magnetic beads. Because the only magnetically non-responsive beadsremaining after Stage 1 separation are those complexed withmagnetically-responsive beads via hybridization to the same contiguousnucleic acid molecule, the number of non-magnetic beads after Stage 1would be proportional to the number of target nucleic acid molecules inthe hybridization mix.

To facilitate separation of the beads by filtration, Stage I could befollowed by detaching DNA from beads (e.g., via DNase treatment orcleavable linker) and filtering the beads through a filter that onlypermits the smaller non-magnetic beads to pass through. The smallerbeads could then be counted using available particle countingtechnologies (e.g., Coulter counter) or quantified using availabletechnologies such as fluorescence, flow cytometry, spectrophotometry,etc. Commercially available beads have a large number of colors andfluorescence wavelengths (e.g., Bangs Labs.).

FIGS. 5, 6, and 7, and Table I provide results that demonstrate thesuccessful separation and quantification of target DNA molecules using acombination of magnetically-responsive and magnetically non-responsivebeads, each type differing in size. FIG. 5 is a photomicrograph of thetwo bead types used in this example. The magnetically-responsive beadsare 2.8 μm diameter and the non-magnetic beads are 0.94 μm diameter(beads are described in Example 1). FIG. 6 shows that all of themagnetically non-responsive beads are eliminated (washed away) duringmagnetic separation if hybridization of the DNA molecule is notperformed to both the magnetic and the non-magnetic beads. In contrast,if hybridization is performed then some of the small beads are pulledalong with the large beads during magnetic separation, as seen on theglass slide in FIG. 7.

If instead of depositing on a glass slide after magnetic separation (aswas done in FIG. 7), the beads are separated from each other bydetaching the hybridized DNA by, e.g., DNase treatment or a detachablelinker between the magnetic bead and the hybridized DNA, and pass thebeads through a 2 μm filter, the 2.8 μm magnetic beads would be toolarge to pass through the filter resulting in a filtered solution ofonly the non-magnetic beads (see data in Table I). These are themagnetically non-responsive beads that were previously hybridized to thesame contiguous piece of DNA as the magnetic beads and thus were pulledalong during magnetic separation. Note that after magnetic andfiltration separation the number of these small beads recovered would beproportional to the number of Type 1+Type 2 DNA in the hybridization mix(this would be true for the present example as well as for target DNAsobtained from a human blood sample). The methods used to obtain theresults in FIGS. 5, 6, and 7, are identical to those described inExample 1. The methods used to obtain the data in Table I are alsoidentical to the methods in Example 1 for DNA target, DNA probes, beads,attachment of probes to beads, hybridization, and magnetic separation.

In Example 2, i.e., where Stage 2 separation is by filtration or sizedistribution analysis, the magnetic separation step in Example 1 isfollowed by detaching the DNA from the beads using DNase treatment(DNase I from Gibco BRL, Frederick, Md.) resulting in the beads becomingfree in solution. One μl of the DNase I stock solution was diluted in405 μl glycerol and 405 μl NTB buffer. The NTB buffer comprises 0.5 MTris-HCl pH 7.5, 0.1 M MgSO₄, 1 mM dithiothreitol, and 500 g/mL bovineserum albumin (Fraction V, Sigma). Then, 22 μl of the diluted DNasesolution was added to 44 μl of the bead solution to be DNA digested(i.e., the beads were in 44 μl 3×NTB). The solution of beads was thenpassed through a filter that permits only the small beads to passthrough, in this example, a filter with 2 μm diameter circular pores(polycarbonate membrane filter available commercially from Millipore,Fisher Scientific Catalog #MP 013 00). Associated syringe filter holdersare also commercially available to perform the filter operation (usedfor the present example, Millipore Stainless Steel 13 Filter Holder,Fisher Scientific Catalog Number XX-30 012 00).

The number of beads in the filtered solution can then be quantifiedusing available methods, e.g., a Coulter counter, microscopy,fluorescence scanning, flow cytometry, spectrophotometry, etc. Apreferred method of bead quantification is by counting in a Coultercounter. At this stage, only the small beads that were hybridized to aType 1+Type 2 DNA are present in the solution, hence the number of thesebeads are proportional to the number of Type 1+Type 2 DNA sequences inthe initial sample (e.g., a blood sample). The proportionality constantcan be obtained from laboratory measurements using standard mixes oftarget DNA and bead-probe complexes. That is, just as routinely done formost kinds of measurement technologies, a standard curve would be usedto convert the measured number of beads to the number of target DNAs inthe original sample.

Table I presents results obtained from a filtration experiment. In thiscase, three different solutions were filtered using the 2.0 μm Milliporefilter system described above: (1) saline only without beads, (2) asolution containing both Dynal and Bangs beads which has been subjectedto the complete hybridization conditions and reagents except that the18.2 kb DNA was not included in the hybridization mix, and (3) asolution containing both Dynal and Bangs beads and hybridized with 20 μgof the 18.2 kb target DNA. The results demonstrate that withouthybridization to the 18.2 kb DNA target molecule, the Bangs beads arenot pulled along with the Dynal beads during magnetic separation. Thatis, without hybridization there is no significant difference between thebead counts and the saline background. This is due to the removal of allBangs beads during the magnetic separation step and subsequent removalof all Dynal beads during the filtration step.

TABLE I Coulter Counter Results for Three Types of Filtered SolutionsMean Counts/100 L SD Filtered Saline 145 109 Filtered beads*, w/o hybrid159 20 Filtered beads**, with hybrid 2910 300Table I shows particle counter results for three types of filteredsolutions, filtered using 2.0 μm Millipore filters (Fisher ScientificCat. No. TTTP 013 00), wherein a Coulter counter Model Zf was used witha 30 μm aperture tube to measure the indicated solutions. The means andSDs are based on multiple measurements.

In contrast, when hybridization is done then the Bangs beads aredetected after filtration. This is due to the fact that both Dynal andBangs beads are hybridized to the same 18.2 kb DNA molecule. Hence, theBangs beads are pulled along by the Dynal beads during the magneticseparation step, released from the Dynal beads by DNase treatment, andfinally separated from the Dynal beads by filtration through a 2.0 mfilter that only permits the Bangs beads to pass through. The resultsfor the conditions of the present experiment show a count signal that is20 times above background, providing a very accurate measurement.

The data in Table I suggest a detection limit for this method of about 5ng target DNA or about 1000 cell equivalents of DNA. This is highlycompetitive with available detection methods (Duggan et al., 21 NatureGenetics (Supplement) 10-14 (1999)).

More generally, this approach can be used to detect rearrangements inany nucleic acid molecule larger than about 10 bp for which suitablehybridization probes are available or obtainable, including DNA at anylevel of organization from single stranded to metaphase chromosomes. Forexample, DNA probes for abl and bcr (which are available) could be usedtogether with the technology described here to rapidly separate andquantify marker chromosomes for human chronic myelogenous leukemia,i.e., so-called Philadelphia chromosomes involving a very specifictranslocation between chromosomes 9 and 22. The abl and bcr genes flankthe fusion point on chromosome 22 (Tkachuk et al., 250 Science 559-562(1990)). To quantify these types of rearranged chromosomes using thepresent invention, a uni-biotinylated probe (only one biotin per probe)homologous to bcr is complexed to the avidin-coated Dynal paramagneticbeads and a uni-biotinylated probe homologous to abl to theavidin-coated Bangs non-magnetic beads and then perform the separationand quantification procedures as described above.

It is also an embodiment of the present invention to employ methods forthe specific detachment of the DNA from the magnetically-responsive beadprior to filtration. If the detachment site was between the DNA and thebead then the complete DNA molecule would remain attached to the smallnon-magnetic bead and would therefore pass through the filter and beisolated together with the small beads. This would permit furtherevaluations/diagnostics of the isolated target DNA. Site-specificdetachment can be accomplished using available cleavable linkers.

Both specific and random rearrangements can be quantified using thisapproach. The 9;22 translocation described above is an example of aspecific rearrangement. An example of the quantification of randomrearrangements is if we selected a probe with unique homology tochromosome 1 (i.e., Probe 1) and a cocktail of composite probes withhomology to other chromosome(s) but not to chromosome 1 (i.e., Probe 2).The more sequences of non-chromosome-1 targets covered by Probe 2, themore translocations would be detectable. Probe 1 would then be complexedwith the 2.8 μm paramagnetic beads and Probe 2 would be complexed withthe 0.94 μm non-magnetic beads. If, in this example, Probe 2 hybridizesto chromosome 1 then interchromosomal rearrangement has in factoccurred. These translocated chromosomes can then be separated andquantified using the method described above. It is expected thatstandard calibration curves would be obtained for each particular kindof detection kit, i.e., one for each specific rearrangement and one fora particular class of random rearrangements.

Size distribution analysis. If, after magnetic separation and DNAdetachment (described above), the resultant solution of beads isanalyzed using a particle sizer/counter (e.g., a Multisizer II byCoulter), the two bead types can be separated by their sizedistributions and the number of beads in each distribution quantified.This is illustrated in FIG. 8. It is seen that the distributions for the4.4 μm beads and the 2.8 μm beads are well separated and that the numberof beads in each distribution can be quantified by integration of thepeaks. In contrast to Table I, the distributions in FIG. 8 show theresults of a DNA separation experiment in which the magneticallynon-responsive beads were larger than the superparamagnetic beads. Inthis case, the larger non-magnetic beads were selected to place the peakin a region of lower background counts. The materials and methods usedto obtain the results in FIG. 8 were the same as those used for Table 1with the following differences: FIG. 8 used 4.4 μm diameter magneticallynon-responsive polystyrene beads coated with streptavidin (Bangs Labs,Fisher, Ind.) whereas Table 1 used 0.94 μm diameter magneticallynon-responsive polystyrene beads coated with streptavidin (Bangs Labs);FIG. 8 used 5 μg of 18.2 kb target DNA whereas Table I used 20 μg of18.2 kb target DNA; partial magnetic separation was done for FIG. 8following DNase treatment to reduce (but not fully eliminate) the numberof superparamagnetic beads to provide a lower background level while atthe same time provide a 2.8 μm peak for comparison/illustrationpurposes; and for FIG. 8 the final bead concentration was dilutedtwo-fold just before generating the bead size distributions using theCoulter Multisizer II. Note that when accounting for the differences inthe amount of target DNA hybridized and final two-fold dilution, theresults of Table 1 and FIG. 8 agree very well, i.e., 2910±300non-magnetic beads were recovered in the experiment reported in Table I,while 322 non-magnetic beads were recovered in the experiment shown inFIG. 8. If the two experiments are normalized by the target DNA (5 μg v.20 μg) hybridized and the two-fold dilution, the result in FIG. 8 of 322non-magnetic beads would become 322×4×2=2576 non-magnetic beads, notsignificantly different from 2910 in Table I.

It should also be noted that the intention of the present invention isnot to be limited by two sizes of beads, but rather that many beadssizes may be used in combination in a more complex and multiplexedsystem of separation and detection.

Example 3

This is an example of a method that can be used to separate and quantifynucleic acids (or chromosomes) with two unique and non-overlappingsequences, both on a contiguous nucleic acid molecule or chromosome. Inthis example, the two different sequences are identified as Type 1 andType 2 nucleic acid sequences.

The method of the present disclosure includes, but is not limited to,the use of two kinds of microbeads. One kind of microbead is responsiveto a magnetic field (e.g., Dynabeads Streptavidin, Product #112.05 fromDynal A/S, Oslo, Norway) and could be coated with biotinylated nucleicacid probes complementary to Type 1 nucleic acid sequences by attachingto the avidin on the surface of the magnetically-responsive beads. Theother kind of microbead is not responsive to a magnetic field (e.g.,streptavidin-coated polystyrene beads from Bangs Labs, Fishers, Ind.)and could be coated with biotinylated nucleic acid probes complementaryto Type 2 nucleic acid sequences by attaching to the avidin on thesurface of the magnetically non-responsive beads. The attachment of thetwo probe types to their respective kinds of beads would be performed inseparate solutions. The magnetically non-responsive beads would also becoated with an electrochemiluminescence (ECL) marker, e.g., ruthenium(II) tris-bipyridine NHS ester. See Blackburn, et al. Clin. Chem. 37(9),1534-1539 (1991) for a description of detection using ECL labels.

In the present invention, the ECL marker would be attached to themagnetically non-responsive beads by, e.g., biotinylating the marker andattaching it to the beads either separately or in competition with thebiotinylated probes. An example of DNAs and coated beads is illustratedin FIG. 9.

After coating the respective kinds of beads and washing away unboundreagents, the coated beads are mixed together in a hybridizationsolution containing the target nucleic acid (e.g., isolated DNA ormetaphase chromosomes). Using hybridization reagents and conditionsavailable in the art (e.g., see Example 1 for isolated DNA; the Examplein U.S. Pat. No. 5,731,153 for metaphase chromosomes; and thehybridization methods in U.S. Pat. No. 5,447,841 for “painting” probes)the result is the hybridization of the probes on the microbeads to theirrespective homologous sequences on the target nucleic acid, whichproduces the hybridized complexes shown in FIG. 9.

The next step is magnetic separation. This is accomplished by firstgently shaking the vial to obtain a homogeneous suspension of beads andthen placing the vial in the Dynal magnet stand (Dynal A/S, Oslo,Norway) for 2 minutes to allow beads to migrate to the side of the tube.This is followed by removing the supernatant by aspiration with apipette while the tube remains in the magnetic stand. The tube is thenremoved from the magnetic stand and fresh buffer is added. Thisseparation step can be repeated and results in the removal of all DNAand beads that are not connected to magnetically-responsive beads via acontiguous target molecule, i.e., only the kinds of complexes shown inFIG. 9 should remain in the sample tube after magnetic separation.

Importantly, following magnetic separation, the only complex with theECL label is in fact the one containing both Type 1 and Type 2 DNA. Thiscould, for example, be a fusion between two different chromosomes suchas those resulting from interchromosomal translocations, or it could beany two uniquely different sequences on a contiguous target nucleic acidmolecule.

The next step is detection by ECL. This would be accomplished by addingvast molar excess of tripropylamine (TPA) to the buffer containing themagnetically separated bead complexes and then placing aliquots of thesolution onto an electrode surface. A low voltage is applied to theelectrode which triggers a cyclical oxidation and reduction reaction ofthe ruthenium metal ion which generates the emission of 620 nm photons.Methods and devices that can be used to detect and quantify the ECLlabel on the non-magnetic beads are described in Blackburn, et al. Clin.Chem. 37(9), 1534-1539 (1991), and also in www.igen.com (i.e., the homepage of IGEN International, Inc., which provides a commerciallyavailable instrument to detect ECL signals). Importantly, by keeping ourmagnetic-non magnetic bead complexes intact following magneticseparation (i.e., no DNase treatment) will permit direct measurementusing IGEN's automatic sample processing system which employs a magnetto immobilize each sample during ECL measurement. The magnetic bead inour complex will serve as the immobilizing particle while the attachednon-magnetic bead will contain the ECL label and hence produce the ECLsignal.

Detection is illustrated in FIG. 9, which includes the Type 1+Type 2 DNAbead complex deposited on the electrode surface. A photon is emitted anddetected by a photomultiplier tube (PMT) or other suitable detectionsystem. Such detection systems are now available commercially (e.g.,IGEN International, Inc.). For a given number of ECL labels per bead(which can be selected) the number of photons is proportional to thenumber of non-magnetic beads, which for a given probe/target nucleicacid protocol, is proportional to the number of contiguous Type 1+Type 2target sequences in the sample.

More generally, this approach can be used to detect rearrangements inany nucleic acid molecule of sufficient size to hybridize one or moremicrobeads to each type of nucleic acid, and for which suitablehybridization probes are available or obtainable, including DNA at anylevel of organization from single stranded to metaphase chromosomes.Presently, commercially available microbeads range in diameter fromabout 50 nm to several mm. Fifty nm is equivalent to the length of about50 bp of the DNA molecule and would probably be near the minimumdistance required to hybridize one bead. Of course, the maximum distancewill simply depend on the size of the target sequence. Alternatively,one could hybridize the magnetically-responsive microbead to the Type 1nucleic acid and hybridize a nucleic acid probe with the ECL labelattached directly to the probe without a non-magnetic bead to the Type 2nucleic acid target. This should permit unique hybridization to targetnucleic acid sequences larger than about 10 bp.

Both specific and random rearrangements can be quantified using thisapproach. For example, DNA probes for abl and bcr (which are available)could be used together with the technology described here to rapidlyseparate and quantify marker chromosomes for human chronic myelogenousleukemia, i.e., so-called Philadelphia chromosomes involving a veryspecific translocation between chromosomes 9 and 22. The abl and bcrgenes flank the fusion point on the Philadelphia chromosome. To quantifythese types of rearranged chromosomes using the present invention, wewould attach the probe homologous to bcr to the superparamagnetic beadsand the probe homologous to abl and the ECL label to the non-magneticbeads and then perform the separation and quantification procedures asdescribed above.

An example of the quantification of random rearrangements is if weselected a probe with unique homology to chromosome #1 (i.e., Probe 1)and one or more probes with homology to any other chromosome but not tochromosome #1 (i.e., Probe 2). Probe 1 would then be complexed with theparamagnetic beads and Probe 2 (which could be a composite of severalprobes, including painting probes) would be complexed with thenon-magnetic beads containing the ECL label. If both Probe 1 and Probe 2are hybridized to the same chromosome then interchromosomalrearrangement has in fact occurred. These translocated chromosomes canthen be separated and quantified using the method described above.Standard calibration curves would be obtained for each particular kindof detection kit, e.g., one for each specific kind of rearrangement andone for a particular class of random rearrangements. For example, ECLintensity vs. rearrangements per cell, could be measured in standardsamples of known rearrangement frequencies using standardized kitformats with demonstrated reproducibility.

Example 4

Avidin-coated magnetically responsive microbeads are complexed withbiotinylated antibodies specific for the M allelic GPA protein (FIG.10). Magnetically non-responsive microbeads are complexed withbiotinylated antibodies specific for the N allelic GPA protein. The twotypes of bead-antibody complexes are then incubated with blooderythrocytes (the attachment of beads and complexing agents can be donein any order as long as they are unique). The resulting products are ofthree types: (1) erythrocytes bearing the M protein complexed to beadscoupled to the anti-M antibody; (2) erythrocytes bearing the N proteincomplexed to bead coupled to the anti-N antibody; and (3) erythrocytesbearing both the M and N proteins complexed to both the beads coupled tothe anti-M antibody and the beads coupled to the anti-N antibody.

Magnetic separation results in washing away all erythrocytes withoutmagnetic bead attachments. Next, the complexing agents are simplydigested with protease and the beads analyzed using the particle sizedistribution using the Multisizer II (Coulter, Inc.). Using differentsize particles for M and N results in a readily quantifiable number ofnon-magnetic particles which would be proportional to the number of MNerythrocytes. Information on GPA mutations and the methods to complexantibodies with the M and N allelic proteins on the surface of human redblood cells is available in Langlois, et al., 236 Science 445-448(1987), and references therein, herein incorporated by reference. Themethods to biotinylate the antibodies and attach them to the avidincoated beads are available to those skilled in the art.

The beads may be freed from the cells by digesting the peptidecomplexing agents with proteinase to remove the beads from the cellmembrane. Then, one may obtain a bead size distribution using theMultisizer II and determine the number of large non-magnetic beads bypeak integration. The number of N beads would be proportional to thenumber of MN cells.

Example 5

Here we describe a new technology that provides a method for efficientlyevaluating for the presence, absence, or amplification of nucleic acidsequences in a sample of nucleic acid. An example of the method isillustrated in FIG. 11.

In this method, the target nucleic acid can be DNA or RNA, singlestranded or double stranded, fully purified, or as chromatin orchromosomes. Preferably, the nucleic acid would be extracted as purifiednucleic acid (e.g., from cells) and evaluated as genomic or fractionatedto segments of sizes, suitable for the particular evaluation beingperformed (e.g. by restriction enzyme digestion). The minimum length ofthe target nucleic acid is on the order of 10 bp (it has to besufficiently long for near unique hybridization). There is no maximumlimit for the length of the target nucleic acid, i.e., it could be thewhole genome.

Materials:

(A) Probes. Nucleic acid probes are made that are complementary tospecific regions of the target nucleic acid to be evaluated. Theindividual probes can be of various lengths (typically 50 bp to 1000 bp)and each probe with two complexing agents, typically incorporated intothe nucleic acid probe by available nick translation methods.

(B) Antibodies. Antibodies are biotinylated and complexed withstreptavidin-coated microspheres. The biotinylation includes a spacerbetween the antibody and the biotin molecule to limit interference withantibody-antigen binding.

(C) Microspheres. Microspheres (typically, 1 to 20 μm diameterpolystyrene beads) are coated with streptavidin. Each bead size would becoated with only one kind of complexing agent.

Methods:

(A) Extract nucleic acids (e.g., from cells) and process as desiredusing available methods.

(B) Insert-peptide-dUTP into probes by available nick translationmethods.

(C) Hybridize the nucleic acid probes to the target nucleic acid insolution and purify to remove unhybridized probes.

(D) In separate solutions, attach the biotinylated antibodies to theirrespective beads (one kind of antibody for each bead size).

(E) Simultaneously, react the coated beads with the target DNA-probecomplexes to attach the beads to their respective complexing agents onthe probes.

(F) Place the solution into a vial coated with a unique complexing agentthat complexes with the probes hybridized to the target DNA. Afterattachment to the vial surface, wash several times to remove unattachedbeads.

(G) Separate the beads from the nucleic acid by protease treatment andquantify size spectrum using a Multisizer II (Coulter, Inc.)

(H) The number of beads of a particular size would be proportional tothe number of target sequences in the nucleic acid sample. Hence, e.g.,gene amplification would result in more beads of a particular size thanexpected from a control sample. Similarly, Downs Syndrome (an extrachromosome 21) would result in 50% more beads attaching to probescomplementary to chromosome 21. In contrast, a deletion of a particulartarget sequence would result in fewer beads than expected. Using manydifferent bead sizes would permit the evaluation of many target nucleicacid sequences simultaneously by using calibration curves, one for eachtarget sequence, to translate the number of beads in each peak into theconcentration of the corresponding target sequence in the sample.

Example 6

In this example, we describe results using our particle analysis assayto rapidly quantify bcr/abl chromosomal fisions in genomic DNA extractedfrom a human chronic myelogenous leukemia cell-line. It is demonstratedthat our assay makes possible very rapid and low cost quantification ofrearrangements in genomic DNA without the need for cell culturing,microscope scoring, or sequence amplification. The assay requires onlyseconds to “score” the number of chromosomal translocations typicallytaking weeks or even months of very costly technician time by availablecytogenetic methods. The principle of the assay is to use the diameterof microparticles as the detectable marker in a sandwich-type assay andthe number of such particles as the quantitative measurement. Theposition of particle attachment to the target DNA can be selected by thesequences of the hybridization probes and the unique complexing agentson the particles. The “scoring” of these detectable markers isaccomplished by automated size-distribution analysis that requires only10 to 15 seconds per sample regardless of how many cell-equivalents arebeing evaluated.

The particle analysis assay was used to detect Ph¹ chromosometranslocations in isolated genomic DNA from a human CML cell line knownto be Ph¹ positive, K-5628, and from a control cell line (H-1395) knownto be Ph¹ negative (American Type Culture Collection, ATCC Cell-LineNumber CRL-5868, http://www.atcc.org; M. R. Speicher et al., 80Laboratory Investigation 1031-1041 (2000)). Commercially-availablepainting probes for the bcr/abl fusion region were used to capture thetarget sequences on the solid support surface (in this case, magneticbeads) and to attach the 5.7 μm diameter non-magnetic particles usedhere for quantification. The probe hybridization cocktail containeddigoxigenin-labeled bcr probe and biotinylated abl probe. Followingsimultaneous hybridization of the probes to the genomic target DNA, thebeads were added to the solution resulting in theanti-digoxigenin-coated magnetic beads complexing with the bcr probesand the streptavidin-coated non-magnetic beads complexing with the ablprobes. This was followed by magnetic separation to remove unattachednon-magnetic beads, DNase treatment to cut the fusion DNA connecting themagnetic and non-magnetic beads, another magnetic separation step toremove the magnetic beads, and then the acquisition of a particle-sizespectrum and counting the number of 5.7 μm particles. The magnetic beadswere removed, in this case, because their size distribution was verybroad and would have interfered with the size distribution of thenon-magnetic particles used here for detection. In practice, removal ofthe magnetic beads would not be required because their size distributionwould be selected to prevent interference with the size distribution ofthe non-magnetic particles. For the present evaluation, these particularmagnetic beads were used simply because they were available commerciallywith anti-digoxigenin coating.

Particle-size spectra obtained using this method are illustrated in FIG.12A-D. The number of particles counted is presented as a function ofparticle diameter. The total number of particles of a particular size(in this case, the observed peak) is obtained automatically byintegration using a computer interface with the particle counter. It isobserved in FIG. 12 that the level of background noise outside the peakis relatively low resulting in a high signal-to-noise ratio. It is alsoobserved that the size of the peak decreases with decreasing target DNA(i.e., decreasing number of bcr/abl fusions) present in the samplesolution. The result in FIG. 12A was obtained using 165 ng genomic DNAfrom the human CML cell line. A single large peak is observed with amean diameter of 5.7 μm. This peak is composed of the non-magneticmicroparticles recovered after hybridization and magnetic separation. InFIG. 12B, a smaller peak is observed at 5.7 μm particle diameter. Thispeak includes the non-magnetic microparticles recovered using 16.5 ngtarget DNA. In FIG. 12C, the hybridization solution contained onlyprobes and beads, but no genomic DNA from the CML cells. In this case,only a very small peak is observed at 5.7 μm. Although small, the factthat a detectable peak exists at all in this size interval indicatesthat some of the microparticles must have become attached to themagnetic beads during the hybridization procedure even though target DNAwas not present. In order to determine how the particles may have becomeattached, we performed an experiment where all conditions were identicalto those in FIG. 12B except that we did not include probes. Theseresults are seen in FIG. 12D and show that the peak in FIG. 12Bdisappeared when the probes were not included. Based on these results,it is clear that the non-unique-sequence “painting” probes used here forabl and bcr exhibit some non-specific inter-probe hybridization, whichresulted in some, albeit small, cross-attachment of magnetic andnon-magnetic beads. Importantly, it is expected that this can beeliminated or at least substantially reduced by using unique-sequenceprobes. The small “background” seen in FIG. 12D from ˜2 μm to about ˜8μm is the residual from the broad size distribution of the magneticbeads used for these experiments. As mentioned above, the magnetic beadswould not be present in the non-magnetic particle interval if they wereselected to have a non-overlapping size distribution.

The numerical results are presented in Table II. These values are theintegrals of the peaks. The genomic K-562 DNA per 500 μl sample analyzedwere 0, 0.165, 1.65, 16.5, and 165 ng. Given that the K-562 cells areessentially triploid8, the number of cell-equivalents per sample rangedfrom 0 to 18,000. Also, based on the frequency of Ph¹ chromosomes inthis cell line8, we estimate that the bcr/abl fusions would range from 0to 2700 per sample. It is observed that there is a clear relationshipbetween the number of beads counted per sample and the number ofexpected bcr/abl fusions in the sample. It appears from these initialresults that on the order of 1 microparticle is counted per bcr/ablfusion in the sample.

TABLE II Quantification of Ph¹ fusions in human genomic DNA. Genomic DNAbcr/abl probe Expected number Number of Counting time per sample # cellper sample of Ph¹ in beads counted per sample (ng) equivalent (μl)sample^(a) per sample^(b) (sec) K-562 cells^(c) 165  18,000 0.5 27001430 ± 38  12.8 16.5 1800 0.5 270 779 ± 28 12.7  1.65 180 0.5 27 151 ±12 12.8   0.165 18 0.5 2.7 138 ± 12 12.7  0^(d) 0 0.5 0 108 ± 10 19.1 16.5^(e) 1800 0 270 49 ± 7 12.6 H-1395 cells^(c) 16.5 2700 0.5 0 58 ± 812.8  0^(d) 0 0.5 0 53 ± 7 12.9 Separated magnetic beads only^(f) 17 ± 412.6 Saline only^(g) 0 12.8 ^(a)Based on 15% Ph¹ chromosome frequency inthe K-562 cell culture used here⁸. ^(b)Mean ± 1 s.d. based on countingstatistics only. ^(c)Cells are described under experimental protocol.^(d)Repeated the procedure as above, but without genomic DNA.^(e)Repeated the procedure as above with 16.5 ng genomic DNA, butwithout probe. ^(f)Magnetic beads were placed in a saline solutionwithout DNA, probes, or non-magnetic beads. The magnetic beads were thenremoved by magnetic separation and the residual number of beads in therelevant interval counted. The initial number of magnetic beads were thesame as in all of the above measurements. ^(g)Pure saline solution wascounted, without any beads or other reagents present.

The results in Table II also include several controls. When K-562 DNAwas not present in the sample, then 108 particles were counted in the5.0-6.8 μm diameter interval. As indicated above, we believe that thesebeads resulted from non-specific hybridization between the commercialpainting probes used here as well as from residual magnetic beads thatwere not fully removed by the second magnetic separation step. Thisconclusion is supported by the 49 beads observed when no probes werepresent in the K-562 experiment suggesting that the probe-probenon-specific hybridization contributed about half of the 108 beadsobserved. We have also observed that the number of residual magneticbeads remaining after magnetic removal can vary somewhat betweenexperiments and can account for a substantial fraction of the 49 beadsobserved without probes. Seventeen beads were observed in the 5.0-6.8 μminterval following the separation of magnetic beads alone, i.e., withoutthe presence of probes, DNA, or non-magnetic beads. These were residualmagnetic beads that remained after the magnetic bead removal step. Takentogether, it appears that the background level for the PCA is on theorder of 100 particles when using commercially available painting probesand the magnetic beads employed here. Given that the background of thecounting instrument itself (using pure saline) is essentially zero inthe relevant size interval, it is expected that substantial reductionsin background are possible by using unique-sequence probes instead ofthe “painting” probes employed in the present experiments and by usingmagnetic beads (or other solid support) that will not interfere with thesize distribution of the non-magnetic particles.

Also presented in Table II are results for a human cell line (H-1395)which does not have Ph¹ chromosomes. Genomic DNA from these cells wasused to measure the non-specific hybridization between the bcr/ablprobes used here and human genomic DNA. In this case, we repeated the16.5 ng DNA measurement by adding H-1395 genomic DNA instead of K-562genomic DNA in the sample. We also included a simultaneous control,i.e., no genomic DNA. The results are clear. Only 58 beads were countedwhen we used H-1395 DNA compared with 779 beads counted when we usedK-562 DNA. This demonstrates that non-specific hybridization isrelatively low and contributes less than 8% of the beads counted with16.5 ng genomic DNA. Given that 53 beads were counted with probe alone,most of this 8% was actually from inter-probe hybridization. This isconsistent with the 3:1 probe-to-genomic DNA in the sample.

The counting times per sample are also listed in Table II. Thesecounting times should be compared with the efforts required formicroscopic analysis of cytogenetic preparations. The sample measuredhere of 165 ng genomic DNA is equivalent to about 18,000 cells. UsingFISH cytogenetics, a technician typically scoring about 200 cells perday would require about 4 months to score 18,000 cells. In contrast, ourbead counting method completed the “scoring” in only 12.8 seconds.Clearly, this dramatic advance in the quantification speed ofchromosomal rearrangements opens new possibilities in medicine, health,and research hitherto not possible due to the inefficiencies andinadequacies of available assays.

Finally, the PCA approach has many other potential applications as well.For example, multiple nucleic acid rearrangements could be quantified inthe same sample simultaneously by selecting beads of various diameters,each diameter representing a particular type of rearrangement. Thiswould result in several peaks in FIG. 13A, each peak with a differentmean particle diameter. Also, random inter-chromosomal rearrangements,such as may be caused by certain environmental agents, could be rapidlyquantified by using a cocktail of whole-chromosome painting probes, eachprobe complementary to a different chromosome. In this case, the largestchromosomes would preferably be used to maximize the fraction of thegenome evaluated. Because of it's intrinsic quantitative nature, the PCAmethod should also be a powerful tool for the detection of more subtlealterations such as deletions, gene amplifications, and a variety ofother genetic abnormalities. We have also shown that the PCA method canbe used to quantify specific proteins. For protein detection, thecomplexing agents are antibodies instead of nucleic acid probes and themicroparticles are used as the detectable marker instead of, forexample, a radioisotope in the case of radio-immunoassays (RIAs). It isinstructive to note that automated particle counting detects 100% of thedetectable marker in the sample in about 13 seconds while scintillationcounting of a radiolabel such as ¹²⁵I would require 40 days (theradiological half life of ¹²⁵I) to detect at most 50% of the marker.Furthermore, as with DNA rearrangements, the PCA approach can be used toquantify multiple proteins simultaneously in a single sample bygenerating a spectrum of different size particles, each size unique fora particular protein.

Experimental Protocol

Target DNA. Genomic DNA from two different human cell lines were used inthese experiments. Purified genomic DNA from the K-562 human chronicmyelogenous leukemia cell-line was purchased from the American TypeCulture Collection (ATCC; Rockville, Md.). These cells have beendetermined to contain an average of about 69 chromosomes per cell andhave a Ph1 chromosome frequency of =15% (Culture B in ref. 8). Purifiedgenomic DNA from the H-1395 human lung adenocarcinoma cell-line was alsopurchased from ATCC. This is a near-normal cell-line without anyapparent Ph1 chromosomes (American Type Culture Collection, ATCCCell-Line Number CRL-5868, http://www.atcc.org; M. R. Speicher et al.,80 Laboratory Investigation 1031-1041 (2000)).

Probes. The m-bcr/abl (minor breakpoint, Catalog Number P5120)translocation DNA probe was obtained from Ventana Medical Systems, Inc.(Tucson, Ariz.). The probe is a mixture of digoxigenin-labeled DNAprobes specific for the minor breakpoint region of the bcr locus onchromosome 22, and biotin-labeled DNA probes specific for the abl locuson chromosome 9. The probes specific for the m-bcr gene are proximal tothe translocation breakpoint on chromosome 22 and the probes specificfor the abl gene are distal to the translocation breakpoint onchromosome 9. The DNA probe solution is premixed with blocking DNA in50% formamide and 2×SSC.

Microparticles. The streptavidin-coated non-magnetic particles (5.7 μmdiameter, s.d.±0.3 μm) were purchased from Bangs Laboratories, Inc.(Fishers, Ind.). These were monodisperse polystyrene microspheressuspended in a stock solution of 9.4×107 beads per mL. Theanti-digoxigenin-coated magnetic particles (about 1 μm mean diameter,but with a broad distribution of sizes) were purchased from RocheMolecular Biochemicals (Indianapolis, Ind.). These weresuperparamagnetic polystyrene particles with no residual magnetism ofthe particles after removal of the magnet. The magnetic particles weresuspended in a stock solution of 1.5×1010 particles per mL.

Hybridization. A 10 μl solution consisting of 3.3-μg genomic DNA in Trisbuffer was added to 40 μl of denature solution with final concentrationof 70% (vol/vol) formamide and 2×SSC. This solution was heated to 70° C.in a water bath for 5 min and then transferred to a 30 μl hybridizationsolution containing 10 μl probe. The final hybridization solutioncontained 50% (vol/vol) formamide, 2×SSC, 1% (vol/vol) salmon sperm DNA(Sigma, St. Louis, Mo.), and 1% (vol/vol) SDS (10% Sodium dodecylsulfate solution, Sigma, St. Louis, Mo.). The mixture was then incubatedwith gentle agitation overnight at 40° C. in a Lab-Line Environ-Shaker.

Particle attachment to probes. After hybridization, 200 μl TE buffer (10mM Tris-HCl, pH 7.5; 1 mM EDTA) and 230 μl B&W buffer (2 M NaCl; 10 mMTris-HCl, pH 7.5; 1 mM EDTA) were added to the sample and then 25 μl ofmagnetic particle solution and 75 μl non-magnetic particle solution wereadded. The magnetic particles were pre-washed with B&W twice and thenon-magnetic particles with B&W/TE (1:1 vol/vol) twice. The mixture wasrotated gently at room temperature for 1 h.

Magnetic separation. The magnetic particles, and those non-magneticparticles cross-linked with magnetic particles via hybridization to thesame contiguous target DNA sequence, were collected with a magneticparticle concentrator (MPC, Dynal, Inc, Lake Success, N.Y.) and thesupernatant was removed. The particles collected were washed once with1×SSC, 0.2% SDS solution and once with 0.1×SSC, 0.2% SDS solution (10min each), followed by washing with B&W three times and TE once, eachwith 200 μl.

Particle counting. The magnetically separated particles were digestedusing DNase I (Gibco BRL, Grand Island, N.Y.) according to themanufacturer's protocol to detach all particles that may be cross-linkedby hybridization. The magnetic beads were collected by MPC and thesupernatant transferred to a counting cuvette (Beckman Coulter, Inc,Miami, Fla.) containing Isoton II diluent (Beckman Coulter, Inc, Miami,Fla.). Final volume was 10 mL. The particles remaining in the solutionwere counted using a Coulter Multisizer II (Beckman Coulter, Inc, Miami,Fla.). The measurement volume was 500 μl.

Example 7

This new technology can be used to quantify many different kinds oftarget molecules simultaneously and can be used to detect any moleculefor which there exists two non-cross reacting complexing agents. Forexample, proteins of importance in health and nutrition such asferritin, transferrin, transferrin receptor, folic acid, vitamin-B12,vitamin-A retinol binding protein, insulin, cortisol, estradiol, FSH,LH, progesterone, T3, T4, and TSH have two commercially availableantibodies that bind to different sites on the same molecule and wouldtherefore be detectable individually or simultaneously using the presentmethod. Antibodies have been developed for a large number of othermolecules as well which can also be quantified using the present method.In addition, modern antibody production can now be used to developlow-cost antibodies against almost any desired target molecule.

The protein separation and quantification technology is illustrated inFIG. 13. For the detection of proteins, one antibody type (preferably amonoclonal antibody) would be attached to a solid support, e.g., to thesurface of a super-paramagnetic microbead (M), and another antibody type(usually a polyclonal antibody attaching to several sites on the sameprotein) attached to the magnetically non-responsive microbeads (N). Inthis example, the antibodies are coated directly onto the surface ofcarboxylic acid coated beads. Other attachment methods are also possiblesuch as biotin/avidin, digoxygenin/anti-digoxygenin, and otherantibody/antigen complexing reactions.

We have successfully performed experiments using our Particle AnalysisAssay to detect protein ferritin. We performed our initial tests usingthis protein because it is very important in the evaluation of ironstatus in humans. Our results of separation and particle sizedistribution analysis are illustrated in FIG. 14. Two peaks are seen,one with a mean particle diameter of 2.8 μm (the super-paramagneticbeads) and another with a mean particle diameter of 4.45 μm (themagnetically non-responsive beads). It is clear that these two peaks areeasily resolved and that a much larger number of peaks could also beresolved. The total number of beads of each kind can be obtained fromthe integral of each peak. In this case, there were a total of 808non-magnetic beads observed in a 0.5 mL aliquot of the 10 mL finalsample solution. This translates into a total of 808×20=16,160non-magnetic beads recovered following the magnetic separation step.Based on a total of 0.27 μg ferritin in the initial test solution, wecalculate a proportionality constant of 3.75×10⁻¹⁷ mole of ferritin pernon-magnetic bead recovered. The minimum detection limit of this methodis only a few beads. For identical protocol conditions, this calibrationconstant could be multiplied by the number of non-magnetic beadsrecovered from an unknown sample of ferritin to obtain the ferritinconcentration in the unknown sample from the number of non-magneticbeads counted in the unknown sample.

Experiments were also performed to measure the number of counts in therelevant size windows when ferritin was not present in the solution.FIG. 15 shows that when ferritin is not included the peak fornon-magnetic beads is not present, demonstrating that there is nodetectable non-specific cross-reactions between beads.

Based on these results, and the fact that we have not yet optimize andfine tuned the method, we estimate the detection limit for this methodto be in the amole range, considerably lower than availableradio-immunoassays and ELISAs, which are typically in the pmole to fmolerange.

Experimental Protocol

Coat beads with ferritin-specific antibodies. Magnetically-responsivebeads (Dynabeads M-270) with carboxylic acid surface coating (Catalog#A143.05) were purchased from Dynal A/S, Oslo, Norway. These were 2.8 μmmean diameter microspheres in 2×10⁹ beads per mL stock solution. SuspendFirst, the Dynabeads M-270 were fully suspended in the stock solution bypipetting and vortexing for 1 min. Immediately pipette 100 μl into a 1.5mL Eppendorf tube. Place the tube in a Dynal magnet stand (MPC) for 4min and remove the supernatant. Resuspend beads in 100 μl of 0.01 MNaOH. Mix well for 5 min and repeat once. Wash beads twice with 100 μlof 0.1 M MES (2-[N-morpholino]ethane sulfonic acid) buffer pH 5.0 andonce 100 μl of cold Milli-Q water. Add 200 μl of 0.005 M CMC(N-cyclohexyl-N-(2-morpholinoethyl)carbodimde methyl-p-toluensulfonate)in cool Mili-Q water to beads and vortex to mix properly. Incubate for10 min at 4° C. with slow tilt rotation. Remove supernatant using magnetstand. Add 120 μl of 0.005 M CMC (Catalog #C1011, Sigma) and 80 μl of0.3 M MES (Catalog #M2933, Sigma). Vortex and incubate as above for 30min. Wash beads twice with cold 200 μl of 0.1 M MES as quickly aspossible. Resuspend Dynabeads M-270 in 150 μl of 10 mM MES containing 60μg of monoclonal anti-ferritin (Catalog #M94157, Fitzgerald IndustriesInternational, Inc, Concord, Mass.). Vortex to ensure good mixing ofprotein and beads. Incubate for 20 min at 4° C. with slow tilt rotation.Add BSA (Sigma) to final concentration 0.1% and incubate as above for 4h. Wash with 120 μl of PBS containing 0.1% BSA and 0.1% Tween 20 (Sigma)four times. Resuspend beads in 200 μl of PBS with 0.1% BSA, 0.1% Tween20, and 0.02% NaN₃ and store at 4° C.

Non-magnetically-responsive beads (carboxylic acid coated polystyrenebeads, Catalog #PC05N, Lot #1193) were purchased from Bangs Labs,Fishers, Ind. These were 4.45 μm mean diameter microspheres in 1.988×10⁹beads per mL stock solution. The protocol for coating the Bangs beadswith antibody was the same as described above for the Dynabeads M-270with the exceptions that the Bangs beads were coated with polyclonalanti-ferritin (Catalog #70-XG50, Fitzgerald Industries International,Inc, Concord, Mass.) and employed centrifugation for the washing stepsinstead of the Dynal magnet stand.

Magnetic separation. Mix well 2 million (2 μl) monoclonal antibodycoated Dynal beads with 0.27 μg ferritin (Catalog #30-AF10, FitzgeraldIndustries International, Inc, Concord, Mass.) in TBST (10 mM Tris, 50mM NaCl, and 0.1% Tween 20, pH 7.5) buffer (total 150 μl). Rotate gentlyat room temperature (r. t.) for 1 h. Wash with 150 μl of TBST threetimes using Dynal magnet stand (4 min each). Mix with 2 million (2 μl)polyclonal coated Bangs beads in 150 μl of TBST. Again, rotate gently atr. t. for 1 h. Wash with B&W 4 times and TE once using Dynal magnetstand (4 min each). Digest with 1 μl Proteinase K (from 23 mg/mL stocksolution, Catalog #P2308, Sigma) in 150 μl of 0.01 M Tris and 0.005 MEDTA at 40° C. for 4 hours. Place tube into Dynal magnet stand (2 min)and transfer supernatant into Beckman-Coulter counting cuvette (Catalog#8320592). Wash the tube 3 times with saline (Isoton II,Beckman-Coulter, Inc.) and then dilute with Isoton II diluent (BeckmanCoulter, Inc.) to 10 mL. The same procedure was followed for the controlexperiment except that ferritin was not added to the solution.

Particle size analysis and bead counting. Obtain particle sizedistribution for the beads that remain in the solution using the CoulterMultisizer II (Beckman-Coulter, Inc) It is seen in FIG. 14 that thenumber of non-magnetic particles is readily obtained and theconcentration of the target protein (ferritin in this example) can bequickly determined from the number of non-magnetic beads using standardcalibration data. For example, the non-magnetic beads counted in FIG. 14result in 3.75×10⁻¹⁷ mole of ferritin per non-magnetic bead recovered.For identical protocol conditions, this calibration constant could bemultiplied by the number of non-magnetic beads recovered from an unknownsample of ferritin to obtain the ferritin concentration in the unknownsample from the number of non-magnetic beads counted in the unknownsample.

Multiple Molecular Detection and Quantification Using the Method of thePresent Invention

Target Molecules (antigens): Ferritin, transferrin receptor, TSH,retinol binding protein, and folic acid. In human bood serum.

Microbeads: Beads of different sizes and with various chemical andphysical functional surfaces are available commercially (e.g. Dynal AS,and Bangs Labs, Inc.). Beads of different diameters can be selected toserve as unique identifiers for each of the five target molecules. Thevarious chemical and physical functional surfaces provide theopportunity for selecting and optimizing bead-antibody immobilizationstrategies. A preferred approach is to use streptavidin-coated beads asthe basic substrate upon which the selected biotinylated antibodies canbe attached.

The magnetically-responsive (M) beads can be Dynabeads M-280, 2.8 μmmean diameter. These beads are commercially available with streptavidinsurface coating from Dynal AS, Oslo, Norway.

The non-magnetic (N) beads can be selected to be five differentdiameters, one diameter for each of the five target molecules. For thisexample, bead diameters in the 4 to 20 μm range, (a much broader rangeof sizes can be used) would be selected to eliminate significant overlapof the peaks. Streptavidin coated beads of these sizes (and almost anyother diameter if special order) are available commercially from BangsLabs, Fishers, Ind. The main selection criterion for bead size is tomake sure that we can adequately resolve the peaks in the measured beadsize distribution. We have performed several experiments with varioussize beads from Bangs Labs that provide assurance that the bead sizesselected here should be resolvable.

Antibodies: The antibodies used in this example would be obtained fromcommercial vendors. Monoclonal and polyclonal antibodies for ferritin,transferrin receptor are available from Fitzgerald IndustriesInternational, Inc, Concord, Mass. A monoclonal antibody for retinolbinding protein is available from Fitzgerald. A polyclonal antibody forretinol binding protein is available from US Biological, Swampscott,Mass. Both monoclonal and polyclonal antibodies for TSH are availablefrom Fitzgerald. Monoclonal and polyclonal antibodies for folic acid arealso available from Fitzgerald.

Biotinylation of Antibodies: A biotin molecule would be conjugated witheach antibody using a “BiotinTag Micro Biotinylation Kit” (B-TAG) fromSigma chemical company. It should be noted that this procedure also addsa 12 atom spacer between the biotin molecule and the antibody tofacilitate protein binding. Briefly, the biotinylated antibodies wouldbe synthesized by adding 10 μL (5 μg/μL) ofBiotinamidocaproate-N-hydroxysulfosuccinimide ester (BAC-SulfoNHS) in0.1 M sodium phosphate buffer, pH 7.2 to 0.1 mL of antibody (10 mg/mL)in same sodium phosphate buffer. Then, reacted for 2 hours with gentleshake at 4° C. The biotinylated antibody will then be purified byapplying the biotinylation reaction mixture to a pretreated Micro-spincolumn G-50 packaged with Sephadex G-50. Spin the column for 2 min at700×g. The purified sample is collected at the bottom of the supporttube. Place the column into another tube and add 0.2 mL PBS (0.01 M, pH7.4). Spin the column for 1 min at 700×g and repeat. Pool the fractionscontaining the purified antibody which is now ready to use.

Immobilizing Antibodies on Beads: To immobilize the biotin-labeledantibodies on the streptavidin coated beads, the beads are first washedwith PBS twice and suspended in the same buffer (0.5 mg beads per mL PBSbuffer). To this solution, we will add 5 μg biotinylated antibody andthen react at room temperature (˜22° C.) for 30 min with gentle mixing.The beads will then be washed three times with PBS and resuspended inthe same buffer for use.

NOTE: The preceding bead and antibody preparations can all be done inadvance, and for commercial applications, would be part of a kit. Theseparation and detection steps listed below can be simplified and somesteps (e.g., incubation) shortened substantially.

Magnetic Separation: React the antibody-coated magnetic beads with thetarget antigen (for this example, with human serum). This isaccomplished as follows: To antigen-containing matrix (0.5 mL), add 0.25mg/0.5 mL (PBS buffer supplemented with 0.2% BSA) magnetic beads, andincubate for 1 hour at room temperature. Collect the beads with amagnetic particle concentrator (MPC) and wash three times with PBS.Resuspend the beads with 0.5 mL of the same buffer. To this solution,add non-magnetic beads coated with the second antibody (0.25 mg/0.5 mL,PBS buffer supplemented with 0.2% BSA) and incubate for 1 hour at roomtemperature. Collect the magnetic beads with MPC and wash 5 times withbuffer. In this procedure, non-magnetic beads are removed by themagnetic washes except those that crosslink with the magnetic beadsthrough antibody-antigen-antibody coupling. The beads are then suspendedin 0.5 mL PBS. Note that separation could also be done using a solidsupport surface other than magnetic beads, e.g., the inside of amicrotiter well, as taught in the body of the patent.

As an example, the separation from human serum of ferritin, transferrinreceptor, TSH, retinol binding protein, and folic acid, is accomplishedusing the magnetic separation procedure described above. In this case,100 μL serum is diluted (1/10) with phosphate buffer saline total 1 mL(pH 7.2 containing 0.5 mL/L Tween 20). To this solution are added Dynalbeads, each with only one of the five kinds of monoclonal antibodiesused in this project (0.25 mg beads/25 μl PBS). The mixture is incubatedfor 1 hour at room temperature with gentle rotation. Collect the beadswith a magnetic particle concentrator (MPC) and wash three times withPBS. The beads are resuspended with 1 mL of the same buffer supplementedwith 0.1% BSA. To this solution are added non-magnetic beads coated witha polyclonal antibody, in this case a unique bead size for each kind ofantibody (0.3 mg beads/0.3 mL), and incubated for 1 hour at roomtemperature. Collect the complexes with MPC and wash with buffer fivetimes. In this procedure, non-magnetic beads are removed by multi-washexcept those that crosslink with the magnetic beads throughantibody-antigen-antibody coupling. The beads are then suspended in 0.5mL PBS. The proteins on beads are then cut by use of proteinase K (15 μLfrom 23 mg/mL stock solution), then diluted with Isoton II diluent(Beckman Coulter Counter) to 10 mL and analyzed using the CoulterMultisizer II. Initially, we will perform the magnetic separation andnon-magnetic bead attachment in two separate steps. This approach willremove essentially all of the non-target proteins from the solution andhence reduce any possible non-specific reactions with the non-magneticbeads used here as our detectable marker.

Simultaneous Quantification of Target Molecules: Determine the targetprotein concentration by counting the number of non-magnetic beads aftermagnetic separation. The quantification of target protein concentrationwill be accomplished by digesting the proteins on the beads usingproteinase K (2.5 μL from 23 mg/mL stock solution), allow to react for30 min, and then dilute with Isoton II diluent (Beckman Coulter Counter)to 10 mL and size/count using the Coulter Multisizer II. Generation of abead size distribution requires only 10 to 15 seconds.

The invention may be embodied in other specific forms without departingfrom its essential characteristics. The described embodiments are to beconsidered in all respects only as illustrative and not restrictive. Thescope of the invention is, therefore, indicated by the appended claimsrather than by the foregoing description. All changes that come withinthe meaning and range of equivalency of the claims are to be embracedwithin their scope.

1. A method for detecting and quantifying a variable chromosometranslocation by identifying and quantifying a nucleic acid containingboth a first nucleotide sequence type and a second nucleotide sequencetype from a mixture of nucleic acids in a sample, wherein the firstnucleotide sequence type and the second nucleotide sequence type arenon-overlapping, and the presence of both indicates the variablechromosome translocation, said method comprising: (a) contacting a firsthybridization probe configured for hybridizing to the first nucleotidesequence type with a magnetically responsive first bead to amagnetically responsive first bead via a first pair of complexing agentsto form a first probe-bead complex, and contacting a secondhybridization probe configured for hybridizing to the second nucleotidesequence type with a magnetically non-responsive second bead to amagnetically non-responsive second bead, which is distinguishable fromthe first bead by size, charge, color, or attachability to a solidsupport, via a second pair of complexing agents to form a secondprobe-bead complex, wherein the first and second hybridization probesare specific to the variable chromosome translocation being detected andquantified; (b) mixing the first probe-bead complex and the secondprobe-bead complex with the sample to form a mixture under conditionssuch that the first hybridization probe hybridizes to the firstnucleotide sequence type and the second hybridization probe hybridizesto the second nucleotide sequence type; (c) separating the firstprobe-bead complex and nucleic acids hybridized to the firsthybridization probe portion thereof by applying magnetic force to themixture and then washing the isolated first probe-bead complex andnucleic acids hybridized to the first hybridization probe portionthereof, thereby obtaining a fraction of nucleic acids comprising thefirst nucleotide sequence type hybridized with the first probe-beadcomplex and nucleic acids comprising both the first nucleotide sequencetype hybridized with the first probe-bead complex and the secondnucleotide sequence type hybridized with the second probe-bead complex;(d) separating the second probe-bead complex and the nucleic acidshybridized to the second hybridization probe portion thereof accordingto the properties of the distinguishable feature of the second bead andwashing to remove nucleic acids not hybridized to the secondhybridization probe, thereby separating the nucleic acids comprisingboth the first nucleotide sequence type hybridized with the firstprobe-bead complex and the second nucleotide sequence type hybridizedwith the second probe-bead complex from the nucleic acids comprisingonly the first nucleotide sequence type hybridized with the firstprobe-bead complex; and (e) detecting and quantifying the nucleic acidscomprising both the first and second nucleotide sequence type bymeasuring and quantifying the second probe-bead complex in the fractionof nucleic acids according to the properties of the distinguishablefeature of the second bead, wherein the number of nucleic acidscomprising both the first and second nucleotide sequences is directlyproportional to the number of variable chromosome translocations.
 2. Amethod for detecting and quantifying a variable chromosome translocationaccording to claim 1, wherein the magnetically responsive first beadsand magnetically non-responsive second beads are of different colors,and the magnetically non-responsive second beads are measured andquantified using flow cytometry.
 3. A method for detecting andquantifying a variable chromosome translocation according to claim 1,wherein the magnetically responsive first beads and magneticallynon-responsive second beads are of different sizes, and the magneticallynon-responsive second beads are measured and quantified using a particlecounter.
 4. A method for detecting and quantifying a variable chromosometranslocation according to claim 1, further comprising the step oftreating the fraction of nucleic acids obtained in the separating step(c) with DNase or RNase to cut the nucleic acid connecting the first andsecond beads.
 5. The method of claim 1, wherein the second bead isnon-fluorescent.
 6. The method of claim 1, wherein the nucleic acidscomprising both the first and second nucleotide sequence type comprisechromosomal rearrangements.
 7. The method of claim 6, wherein the firstnucleotide sequence type is from a first chromosome and the secondnucleotide sequence type is from a second chromosome.
 8. The method ofclaim 5, wherein the non-fluorescent bead is tagged with a detectablemarker.
 9. The method of claim 8, wherein the detectable marker isfluorescent.
 10. The method of claim 1, wherein the second bead iselectrically responsive.
 11. The method of claim 1, wherein the secondbead is coated with a complexing agent.
 12. The method of claim 1,wherein the second bead is a different size than the first bead.